Mycobacterium abscessus ESX-3 plays an important role in host inflammatory and pathological responses during infection

Accepted Manuscript Mycobacterium abscessus ESX-3 plays an important role in host inflammatory and pathological responses during infection Yi Sak Kim, Chul-Su Yang, Loi T. Nguyen, Jin Kyung Kim, Hyo Sun Jin, Jin ho Choe, Soo Yeon Kim, Hye-Mi Lee, Mingyu Jung, Jin-Man Kim, Myung Hee Kim, EunKyeong Jo, Ji-Chan Jang PII:

S1286-4579(16)30123-X

DOI:

10.1016/j.micinf.2016.09.001

Reference:

MICINF 4426

To appear in:

Microbes and Infection

Received Date: 25 February 2016 Revised Date:

2 August 2016

Accepted Date: 5 September 2016

Please cite this article as: Y.S. Kim, C.-S. Yang, L.T. Nguyen, J.K. Kim, H.S. Jin, J.h. Choe, S.Y. Kim, H.-M. Lee, M. Jung, J.-M. Kim, M.H. Kim, E.-K. Jo, J.-C. Jang, Mycobacterium abscessus ESX-3 plays an important role in host inflammatory and pathological responses during infection, Microbes and Infection (2016), doi: 10.1016/j.micinf.2016.09.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Mycobacterium abscessus ESX-3 plays an important role in host inflammatory

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and pathological responses during infection

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Yi Sak Kim,1,3† Chul-Su Yang,4† Loi T. Nguyen,5 Jin Kyung Kim,1,3 Hyo Sun Jin,1,3 Jin

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ho Choe,1,3 Soo Yeon Kim,1,3 Hye-Mi Lee,1,3 Mingyu Jung,2,3 Jin-Man Kim,2,3 Myung Hee

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Kim,5 Eun-Kyeong Jo,1,3 and Ji-Chan Jang1,3,6*

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Department of Microbiology and 2Pathology, 3Department of Medical Science, College of

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Medicine, Chungnam National University, Daejeon 301-747, Korea.

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Department of

Molecular and Life Science, Hanyang University, Ansan 426-791, Korea. 5Infection and

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Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology,

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Daejeon 305-806, Korea. 6Molecular Mechanism of Antibiotics, Division of Life Science,

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Research Institute of Life Science, Gyeongsang National University, Jinju, Gyeongnam,

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Korea

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*For correspondence. E-mail: [email protected];

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Tel. (+82) (0)55 772 1368

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† Both authors contributed equally to this work.

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Abstract Mycobacterial ESX systems are often related to pathogenesis during infection.

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However, little is known about the function of ESX systems of Mycobacterium abscessus

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(Mab). This study focuses on the Mab ESX-3 cluster, which contains major genes such as

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esxH (Rv0288, low molecular weight protein antigen 7; CFP-7) and esxG (Rv0287, ESAT-6

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like protein). An esx-3 (MAB 2224c-2234c)-deletional mutant of Mab (∆esx) was constructed

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and used to infect murine and human macrophages. We then investigated whether Mab

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∆esx modulated innate host immune responses in macrophages. Mab ∆esx infection

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resulted in less pathological and inflammatory responses. Additionally, ∆esx resulted in

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significantly decreased activation of inflammatory signaling and cytokine production in

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macrophages compared to WT. Moreover, recombinant EsxG▪EsxH (rEsxGH) proteins

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encoded by the ESX-3 region showed synergistic enhancement of inflammatory cytokine

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generation in macrophages infected with ∆esx. Taken together, our data suggest that Mab

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ESX-3 plays an important role in inflammatory and pathological responses during Mab

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infection.

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Keywords : Mycobacterium abscessus; ESX-3; host inflammation

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1. Introduction Mycobacterium abscessus (hereafter referred to as Mab) is the most pathogenic strain

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of the Mab complex, a group of rapidly growing non-tuberculous mycobacteria (NTM) and a

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pathogenic mycobacterium [1]. Mab is commonly associated with a wide spectrum of human

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diseases, including respiratory tract infections, traumatic skin and soft tissue infections,

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bacteremia, and other infections involving almost all human organs [2,3,4]. Innate immunity

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is a critical host defense system against mycobacterial infection that detects a variety of

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mycobacterial-derived molecular patterns while inducing proinflammatory mediators and

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antimicrobial proteins [5,6,7]. Numerous pattern-recognition receptors, such as Toll-like

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receptors (TLRs), are involved in the recognition of mycobacterial products. These

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mechanisms also trigger complex intracellular signaling activation, culminating in the

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activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling

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in innate immune cells [8,9]. Activation of innate immune signaling leads to the production of

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proinflammatory cytokines, including tumor necrosis factor TNF-α, interleukin (IL)-6, IL-1β,

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and IL-12 [8,10].

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The development of molecular genetic approaches, such as whole-genome sequencing,

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has markedly enhanced our understanding of various properties of Mab [11]. The

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identification and immunological characterization of essential Mab genes contributes to the

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development of novel protective and therapeutic strategies against Mab infection. However,

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little is known about the function of many Mab genes in terms of their interactions with host

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immune cells that elicit innate immune responses during Mab infection. In Mycobacterium

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tuberculosis (Mtb), a widely characterized pathogen, five type VII secretion systems are

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responsible for exporting proteins and members of the ESX family, which are involved in

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tuberculosis pathogenesis and survival within host cells [12,13,14]. The early secreted

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antigenic target of 6 kDa (ESAT-6)/culture filtrate protein of 10 kDa (CFP-10) complex,

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secreted by the ESX-1 secretion system (also known as the RD1 region), plays an essential

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ACCEPTED MANUSCRIPT role in pathogenesis and immune activation during Mtb infection [15]. Furthermore, the ESX-

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5 secretion system of pathogenic mycobacteria modulates the macrophage response in M.

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marinum [16]. Compared to the ESX genomic location in Mtb, much less is known about the

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immunological functions of the Mab ESX region. Mab only has two conserved ESX gene

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clusters (ESX-3 and ESX-4) [17,18]. ESX-3 is conserved in all mycobacterial species [19]

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and has a role in promoting mycobacterial virulence [20]. ESX-3 is also an essential

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secretion system for iron and zinc homeostasis in M. tuberculosis and is consequently linked

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to adaptations of M. tuberculosis in low zinc environments [21].Although this locus has been

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implicated in mycobacterial growth [19], the function of ESX-3 has not been widely studied in

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terms of host–pathogen interaction during Mab infection. Mab includes EsxG and EsxH

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proteins, major proteins encoded by the esx-3 region, which show a high level of homology

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with EsxG and EsxH from Mtb. The present study reports evidence showing an important

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role for Mab ESX-3 in the induction of host immunopathological responses and increased

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mycobacterial growth during infection.

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2. Material and Methods

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2.1. Inactivation of esx gene cluster The mutation construct for the ESX-3 gene cluster was generated by PCR-

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amplification from Mab ATCC 19977 genomic DNA as a template. The primer pair pJV531F

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(CCCAAGCTTAGACCACTTCGCGGGCGACGG) and pJV531R (CTACCTGCAGCACTTAC

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AGCCCTTCACCCG), including HindIII and PstI sites, respectively, was used to amplify

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fragment 1, which consists of N-terminal amino acids together with a region ∼925 bp

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downstream of Mab 2234c. Similarly, the primers pJV532F (CTACCTGCAGGTGCTGGG

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GCGAGCACTTGC)

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containing PstI and XbaI sites, respectively, were used for amplification of fragment 2 (1045

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bp), which includes 140 bp of Mab 2224c. In more detail, fragment 1 was subcloned into

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HindIII/PstI-digested pBluescript II SK(+) and fragment 2 was subsequently subcloned into

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PstI/XbaI-digested pBluescript II SK(+) harboring fragment 1 to generate pBSK-∆esx. The

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zeocin

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(CTACCTGCAGCGCTAGCTCGAGCACGTGTTGACAATTAATCATCGGCATAGTATATC)

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and ZeoR4pcD (CTAGCTGCAGATCTCGTAGCACGTGTCAGTC) from pcDNA3.1/Zeo(+). It

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was digested with PstI, purified, and cloned into the PstI site of pBSK-∆esx to generate

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pBSK-∆esx::zeo. Allelic-exchange substrate (AES) was amplified from pBSK-∆esx::zeo and

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used to perform mutagenesis in Mab. The strain containing pJV53 was cultured and

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incubated with 0.2% acetamide. Electrocompetent cells were prepared with ice-cold 10%

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glycerol solution. The competent cells were electroporated with 100 ng of AES and plated on

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50 µg/mL zeocin after 24 h of incubation at 37 °C. Muta tion events were verified by PCR and

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sequencing.

(CGTCTAGAAGTGCCCTGCCCTCGCACGTC),

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2.2. Bacteria culture condition, enumeration and In vitro growth kinetics

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In vivo examinations were performed with Mab ATCC 19977; Wild-type (WT) and

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∆esx. Both strains were grown for 4 days, at 37°C, in M iddlebrook 7H9 broth supplemented

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with albumin-dextrose-catalase (ADC, Difco) with daily agitation. These cultures were further

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diluted serially (10-fold) to quantify colony-forming units (CFU)/mL.

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spread on Middlebrook 7H10 agar media supplemented with 10% OADC (oleic acid, albumin,

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dextrose and catalase, Difco) and incubated as described above until colonies were visible.

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The visible Mab colonies were counted and adjusted based on the dilution factor. According

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to this CFU determination, stocks of 1 × 108 CFU/mL were prepared and stored at -80°C until

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used. Prior to infection, frozen stocks were thawed and 10-fold serial dilutions were prepared

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in PBS plus 0.05% Tween-80 (PBST) before infection. To determine whether esx-3 gene

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cluster disruption has brought in any change in the in vitro cultivation, we compared the

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growth rates of Mab WT or ∆esx under enriched Middlebrook 7H9 broth supplemented with

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10% ADC. A growth assay was performed in 3 mL shaking cultures at 37°C. The OD600 was

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measured every 12 h for 4 days. All the experiments were performed in triplicate and

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standard deviations were determined.

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2.3. Mice and cells

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100 µL aliquots were

Wild-type female C57BL/6 mice (aged 6–8 weeks) were purchased from SAMTAKO

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BIO (Gyeonggi-do, Korea) and were maintained under specific pathogen-free conditions. All

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animal-related procedures were reviewed and approved by the Institutional Animal Care and

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Use Committee, Chungnam National University School of Medicine (CNUH-014-A0008;

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Daejeon, Korea). All animal procedures were conducted in accordance with guidelines of the

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Korean Food and Drug Administration (KFDA). Murine bone marrow-derived macrophages

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(BMDMs) were isolated and differentiated by growth for 4–5 days in medium containing

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macrophage colony-stimulating factor (25 ng/mL; R&D Systems, 416-ML). The culture

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medium was Dulbecco’s modified Eagle’s medium (DMEM; Lonza, 12-604F, NJ, USA),

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which contained 10% FBS (Lonza, BW14-503E) and penicillin–streptomycin–amphotericin B

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(Lonza, 17-745E). Human monocyte derived macrophages (MDMs) were prepared as

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described previously [22]. Briefly, human monocytes were isolated from blood obtained from

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healthy volunteers. The buffy coat was collected using a Ficoll gradient and monocytes were

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enriched by adherence to tissue culture plastic. The Institutional Research and Ethics

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Committee at Chungnam National University Hospital approved this study.

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2.4. Infection macrophages, mouse infection, histopathology, and bacterial counts

For infection, Mab WT and ∆esx were added to macrophages at 1:1 or 1:10

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multiplicity of infection (MOI). After 4h of infection, extracellular mycobacteria were washed

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out and infected macrophages were maintained in culture medium at 37°C and 5% CO 2 for 3

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days. For bacterial burdens in the spleen and liver, mice per group were intravenously

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infected with 1×107 CFU/mouse of Mab WT or ∆esx. Lungs, livers, and spleens were

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harvested, and homogenates were counted at different times (1, 7, and 14 days) after

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infection. For bacterial counting, the number of viable bacteria in each organ was determined

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by plating serial dilutions of whole-organ homogenates on Middlebrook 7H10 agar

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supplemented with OADC (Difco, Detroit, MI, USA). Colonies were counted after 3–5 days of

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incubation at 37 °C, and the results were calculate d as the mean log10 CFU per organ.

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Control mice were injected with PBST. For secretion of serum cytokine levels, 1×107 bacilli of

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Mab WT or ∆esx were intravenously infected. ELISA was used to evaluate the levels of pro-

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inflammatory cytokines, such as TNF-α and IL-6, in the infected lungs of mice within one day

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post-infection. For IHC staining findings (for lung), groups of five mice each were

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anaesthetized, their trachea exposed via a small midline incision. Each mice were infected

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intratracheally with 5×105 CFU of Mab WT or ∆esx per mouse in 50 µL PBST. Lung samples

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were fixed in 10% formalin and embedded in paraffin wax. Sections were then cut and

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stained with hematoxylin and eosin. Inflammation in lung sections was graded for severity by

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scanning multiple random fields in three sections of each tissue per mouse. An overall

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histopathological score was assigned to each tissue in each animal based on the extent of

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granulomatous inflammation as follows: 0 = no lesion, 1 = minimal lesion (1–10% of tissue in

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section involved), 2 = mild lesion (11–30% involved), 3 = moderate lesion (30–50% involved),

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4 = marked lesion (50–80% involved), and 5 = severe lesion (> 80% involved), as described

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previously [20].

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2.5. Reagents and antibodies Ultrapure lipopolysaccharide (LPS; tlrl-3pelps) was purchased from InvivoGen (San

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Diego, USA). DAPI was purchased from Sigma. For western blotting, specific antibodies

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(Abs) against phospho-SAPK/JNK (4668), phospho-p44/42 MAPK (ERK1/2) (9101), and

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phospho-p38 MAP Kinase (9211) were obtained from Cell Signaling Technology (Beverly,

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MA, USA). Anti-IκB-α (sc-371) and anti-actin (sc-1616) were obtained from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). For IHC staining, paraffin wax-embedded lung

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sections were stained using anti-COX2 (ab15191), anti-neutrophil (ab2557), and anti-iNOS

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(ab3523) Abs biotin-conjugated rabbit anti-mouse IgG, as well as a peroxidase-conjugated

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streptavidin Ab.

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2.6. RNA extraction, quantitative real-time PCR, enzyme-linked immunosorbent assays

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(ELISA) and Western blotting

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Total RNA was extracted from cells using the TRIzol reagent (Thermo Fisher

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Scientific, 15596-026) according to the manufacturer’s protocol. Real-time PCR reactions

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were performed according to the manufacturer’s instructions (SYBR green PCR master mix,

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Qiagen), and thermal cycling was performed in a Rotor Gene 6000 instrument (Qiagen).

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Primer sequences were as follows: mTNFα (forward: AGCACAGAAAGCATGAT

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CCG, reverse: CTGATGAGAGGGAGGCCATT), mIL6 (forward: ACAAAGCCAGAGTCCTTC

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AGA, reverse: TGGTCCTTAGCCACTCCTTC), mIL1β (forward: TGACGGACCCCAAAAGAT

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GA, reverse: AAAGACACAGGTAGCTGCCA), mIL12p40 (forward: AGGTCACACTGGACCA

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AAGG, reverse: TGGTTTGATGATGTCCCTGA), mβactin (forward: CCACCATGTACCCAGG

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CATT, reverse: AGGGTGTAAAACGCAGCTCA), hTNFα (forward: GGCGTGGAGCTGAGAG

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ATAAC, reverse: GGTGTGGGTGAGGAGCACAT), hIL6 (forward: TGTGAAAGCAGCAAAG

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ACCEPTED MANUSCRIPT AGGCACTG, reverse: ACAGCTCTGGCTTGTTCCTCACTA), and hβactin (forward: CACCAT

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TGGCAATGAGCGGTTC, reverse: AGGTCTTTGCGGATGTCCACGT). In the sandwich

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ELISA, serum and cell culture supernatants were analyzed using a Mouse BD OptEIA Set

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ELISA Kit (BD Biosciences, USA) to detect TNF-α (558534), IL-6 (555240), IL-1β (559603),

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and IL-12p40 (555165). The Human Ready-SET-Go ELISA kit (eBioscience, USA) was used

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to detect TNF-α (88-7346) and IL-6 (88-7066). The limit of detection of the assay indicated

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by the manufacturer (BD OptEIA Set ELISA Kit) was mouse 15 pg/ml (for mouse TNF-α), 4

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pg/ml (for mouse IL-6, IL-1β, IL-12p40, and human TNF-α) and 2 pg/ml (for human IL-6). For

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Western blotting, cell lysis was performed with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM

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NaCl, 0.1% SDS, 1% NP-40, 0.5% deoxycholate and protease inhibitors) at 4 °C for 60 min.

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The protein extracts were boiled in SDS sample buffer, loaded onto a 12% SDS-

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polyacrylamide gel for electrophoresis, and transferred to a polyvinylidene difluoride

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membrane (Millipore, IPVH00010). Signals were visualized with ECL solution (Millipore,

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WBKL S0500) and detected with an UVitec Alliance mini-chemiluminescence device (UVitec,

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Rugby, UK). The ImageJ software was used for densitometric analysis of the blots.

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2.7. Immunofluorescence microscopy of NF-κB p65 translocation Translocation

of

NF-κBp65

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detected

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immunofluorescence staining. Briefly, cells were infected with Mab WT or ∆esx for 30 min

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and then fixed with 4% paraformaldehyde in PBS for 10 min. Cells were permeabilized with

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0.25% Triton X-100 in PBS for 10 min and stained with anti-mouse NF-κB p65 (1:400, for 18

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h at 4 °C; sc-372, Santa Cruz Biotechnology, Inc.) and anti-rabbit AlexaFluro 488 (1:400, for

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2 h; A-11008, Invitrogen) at room temperature (RT). Nuclei were stained by incubation with

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DAPI (Sigma) for 2 min. After mounting, fluorescence images were acquired using a

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confocal laser-scanning microscope (LSM 710; Carl Zeiss, Jena, Germany).

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2.8. NF-κB Luciferase reporter assays

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Iowa City, IA, USA) was performed for 36 h, followed by infection with Mab WT or ∆esx for 6

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h. Infected cells were washed three times in PBS, and cell extracts were prepared by adding

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100 µL of 1× Passive Reporter Lysis Buffer (Promega, Madison, WI, USA). Luciferase

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activity was measured using the Luciferase Assay System (Promega) according to the

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manufacturer’s instructions.

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2.9. Recombinant protein purification

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The coding regions for EsxG (MAB_2229c) and EsxH (MAB_2228c) were subcloned

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into NcoI and XhoI restriction sites of the pHis-parallel1 and pGST-parallel1 vectors,

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respectively, containing recombinant TEV protease (rTEV) cleavage sites [24]. ClearColi

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BL21 (DE3) cells (Lucigen, Middleton, USA) harboring each plasmid were grown in LB

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medium containing ampicillin at 37 °C until they re ached an OD600 of 0.4–0.5. After cooling to

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18 °C, protein expression was induced with 0.5 mM I PTG overnight. The cells expressing

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each protein were then harvested and re-suspended in cooled buffer (50 mM Tris-HCl, pH

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8.0, 300 mM NaCl, and 5% glycerol). The cell suspension was lysed with an ultra-high-

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pressure homogenizer (UHPH; BEE International, USA). The expressed EsxG and EsxH

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proteins were then purified by Ni-NTA agarose (Invitrogen, Carlsbad, CA) and GST agarose

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(GE Healthcare, USA) affinity chromatography, respectively. GST-fused EsxH was further

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purified by treatment with rTEV protease (Invitrogen) to remove GST and additional GST-

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affinity chromatography. A complex of purified His-tagged EsxG and tag-free EsxH proteins

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spontaneously formed upon mixing in a ratio of one to one. This complex was subjected to

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size-exclusion chromatography for further purification. The complex protein was analyzed by

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SDS-PAGE followed by Coomassie staining. Removal of endotoxin from recombinant

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proteins was performed using polymyxin B-agarose (Sigma, USA) according to the

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manufacturer’s protocol. Contamination with endotoxin was assessed with the Limulus

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Amebocyte Lysate (LAL) QCL-1000 assay (Lonza, Walkersville, USA).

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2.10. Statistical Analyses All data obtained from independent experiments were analyzed by two-way analysis

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of variance (ANOVA) with Bonferroni post-tests or one-way ANOVA with Bonferroni’s multiple

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comparison test. Results are presented as the mean and standard error of the mean (SEM).

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Differences were considered statistically significant when P < 0.05.

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3. Results

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3.1. Construction of Mab mutants using a recombineering system

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To study the functional role of ESX-3, recombineering was used to replace structural

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gene regions with a zeocin cassette to provide resistance to zeocin [25]. Schematic

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representations of the ESX-3 region genes in Mab WT and ∆esx mutant are shown in

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Supplementary figure 1A and 1B, respectively. The resulting ∆esx strain was confirmed by

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PCR using primers for the genomic region outside of the ESX-3 flanking regions. PCR

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products (expected size 2.4 kb) would only be obtained if the zeocin cassette had been

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inserted into the correct location on the chromosome. The expected PCR product size was

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obtained from putative ∆esx; no product was obtained for WT Mab ATCC 19977 (13.7 kb;

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Supplementary figure 1C). The amplicon was further analyzed by sequencing, which

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confirmed the clear deletion of esx-3 regions from the genome (data not shown).

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Furthermore, individual gene disruption was validated by PCR using individual gene-specific

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primers (Supplementary figure 1D).

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3.2. Esx-3 locus is required for pathological changes during the acute phase of Mab infection

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To determine whether esx-3 gene cluster disruption has brought in any change in the

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in vitro cultivation, we compared the growth rates of Mab WT or ∆esx. Differences in growth

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rate were compared using a nonparametric t test. As shown in Fig. 1A, the ∆esx did not

ACCEPTED MANUSCRIPT displayed a significantly reduced growth rate and doubling time compared to that of the Mab

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WT. The mutant reached a stationary phase at 72 hours after inoculation with a similar

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density compared to the WT (Fig. 1A, P=0.6270). Based on similarity of growth patterns

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between Mab WT and ∆esx, we further assessed the role of esx-3 in Mab survival inside

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macrophages. For this, survival of ∆esx was studied in BMDMs and compared with that of

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Mab WT. The results showed that within 3 days, BMDMs were unable to eliminate the Mab

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WT strain. As shown in Fig.1B, intracellular growth of Mab WT was significantly increased in

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BMDMs compared with ∆esx at 3 days after infection. Similar findings were observed in data

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from different MOIs (Fig.1B), suggesting that esx-3 is required for intracellular survival of

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Mab. This finding was further studied using an in vivo acute model of infection. At days 7 and

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14, the bacterial load in the spleen and liver was determined. As shown in Fig.1C,

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significantly decreased splenic and hepatic bacillary loads were found in mice infected with

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∆esx on day 7 after infection compared with those from mice infected with the WT strain. At

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14 days post-infection, there were no change for bacterial growth in the livers of ∆esx

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infected mice (Fig.1C). The results presented above show that growth of the ∆esx strain was

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inhibited in mouse organs.

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To this end, we investigated whether mice infected with ∆esx and Mab WT strains

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show different pathological responses. As shown in Fig.1D, the lungs of mice infected with

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Mab WT exhibited extensive pathological changes at 7 days post-infection. In more detail,

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the lung sections of mice infected with Mab WT exhibited severe lung pathology

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characterized by much more dense alveolar spaces and increased granulomatous infiltrate.

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However, significantly less granulomatous infiltrate was observed in ∆esx-infected animals.

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Quantitative scoring of histopathological changes confirmed that the Mab WT group (n=5)

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showed much more severe lung pathology than the ∆esx group at 7 days post-infection

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(Fig.1D). Together, these pathological changes demonstrate that esx-3 influences the

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pathogenesis of Mab in mice.

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3.3. Mab ∆esx infection induces less inflammatory and pathological responses in vivo Mab vigorously activates innate immune responses in macrophages through

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interactions between TLR2 and dectin-1 [26]. However, the role of the Mab esx-3 locus is not

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well known in mice or macrophages. The serum levels of TNF-α and IL-6 in C57BL/6 mice

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after infection were analyzed to examine if mice infected with ∆esx produced a more

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attenuated inflammatory response than those infected with Mab WT. As shown in Fig.2A,

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∆esx elicited a reduced amount of both TNF-α and IL-6 compared to the WT strain,

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especially at one day post-infection.

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Expression of cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), and

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neutrophils in lungs was further evaluated by immunohistochemistry. Immunoreactivity

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against three antibodies (COX-2, iNOS, and neutrophil) was investigated in lung tissues

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infected with Mab WT or ∆esx strains (Fig.2B and 2C). The amount of positively stained cells

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was evaluated to calculate the percent of positive cells for scoring. As shown in Fig.2B,

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COX-2, iNOS, and neutrophil staining in lung sections differed significantly between the

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infected strains. Mab WT infected lung tissue samples showed >58% COX-2 and >79%

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iNOS positive expression rates in immunohistochemical scoring (Fig.2C). In contrast, ∆esx

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infected lung tissues showed lower positive rates for COX-2 (25%) and iNOS (52%)

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expression. In addition, ∆esx infected lung tissues had lower rates of neutrophil infiltration

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compared to those infected with Mab WT (Fig.2B and 2C). Together, these data suggest that

331

systemic and local inflammatory responses are markedly reduced in ∆esx-infected mice

332

compared with those of Mab WT-infected mice.

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3.4. Mab ∆esx leads to less proinflammatory cytokine production in murine and human

335

macrophages compared to WT strain

336

The contribution of the ESX-3 region to pro-inflammatory responses was further

337

examined in murine and human macrophages. Proinflammatory cytokine generation was

ACCEPTED MANUSCRIPT compared for Mab WT- and ∆esx-infected BMDMs as well as human monocyte-derived

339

macrophages (MDMs). Mab WT robustly induced mRNA expression of proinflammatory

340

cytokines (TNF-α, IL-6, IL-1β, and IL-12 p40) from BMDMs after 3 h of infection. The mRNA

341

levels of proinflammatory cytokines induced by ∆esx were significantly lower than those

342

induced by Mab WT at different time points, as shown in Fig.3A. BMDMs infected with ∆esx

343

also produced smaller amounts of proinflammatory cytokines after 18 or 48 h of infection

344

compared to those infected with the Mab WT strain (Fig.3B).

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The mRNA and protein expression of proinflammatory cytokines in human MDMs was

347

further examined after Mab WT or ∆esx infection. Similar to findings observed in murine

348

BMDMs, human MDMs tend to produce less TNF-α and IL-6 mRNA at different time points

349

(3 and 6 h for TNF-α; 6 and 18 h for IL-6; Fig.3C) in response to ∆esx than in response to

350

the WT strain. In addition, ∆esx infection produced smaller amounts of TNF-α and IL-6 in

351

MDMs at different time points (from 6 to 18 h for TNF-α; from 6 to 48 h for IL-6) compared to

352

Mab WT infection (Fig.3D). These results demonstrate that the ESX-3 region of Mab plays a

353

role in the induction of inflammatory responses in murine and human macrophages.

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3.5. Mab ∆esx significantly attenuates activation of MAPK signaling in macrophages

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The MAPK pathways play a key role in the host inflammatory response during

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mycobacterial infection [27,28]. To examine the molecular mechanisms underlying ESX-3-

358

mediated inflammatory responses, the activation of ERK, p38, and JNK MAPKs in BMDMs

359

infected with either Mab WT or ∆esx was examined. As shown in Fig.4A, Mab WT robustly

360

activated three families of MAPKs in BMDMs after infection in a time-dependent manner. As

361

expected, the ∆esx-infected BMDMs showed less phosphorylation of ERK, p38, and JNK

362

MAPKs at different time points compared to WT-infected BMDMs (Fig.4A). Densitometric

363

analysis showed that the phosphorylation levels of ERK, p38, and JNK MAPKs were

364

significantly decreased in BMDMs infected with ∆esx at 15 to 60 mins post-infection

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compared to those infected with the WT strain (Fig.4B). Moreover, the ∆esx-induced

366

phosphorylation of ERK, p38, and JNK MAPKs was significantly decreased in BMDMs

367

compared with those infected by WT (Fig.4C). Taken together, these results suggest that the

368

ESX-3 region is associated with MAPK activation in BMDMs after infection.

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3.6. ESX-3 locus contributes to the activation of NF-κB signaling in macrophages during Mab

371

infection

We next examined whether the ESX-3 locus was involved in nuclear translocation and

373

activation of NF-κB, an essential transcriptional factor in inflammatory signaling [29], after

374

infection with ∆esx. Subcellular localization of NF-κB p65 in the nucleus was significantly

375

reduced in BMDMs infected with ∆esx compared to those infected with WT, as determined

376

by confocal microscopy after 30 min of infection (Fig.5A). This observation was further

377

analyzed by quantifying the effects of Mab WT and ∆esx on translocation of NF-κB/p65.

378

Quantification of cells from different fields showed a significantly lower percentage of NF-

379

κB/p65 translocation into nuclei in BMDMs infected with ∆esx compared with Mab WT

380

infection. Moreover, this relationship operated in a MOI-dependent manner (Fig.5B).

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A NF-κB luciferase assay was also performed in BMDMs transduced with adenovirus

383

encoding a luciferase reporter plasmid containing response elements for NF-κB (Ad-NF-κB-

384

Luc). The activity of the NF-κB reporter gene was considerably reduced in BMDMs

385

transduced with Ad-NF-κB-Luc with ∆esx infection compared to those infected by Mab WT

386

(Fig.5C). As shown in Fig.5D, the effect of ∆esx on IκB-α degradation in BMDMs was also

387

examined. Degradation of IκB-α was prominent in BMDMs after 15-30 min of Mab WT

388

infection. However, few changes in IκB-α degradation were found in BMDMs after ∆esx

389

infection. Thus, the ESX-3 region enhanced activation of NF-κB signaling in BMDMs during

390

Mab infection.

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3.7. rEsxGH protein is required for synergistic synthesis of TNF-α and IL-6 in BMDMs

393

infected with Mab ∆esx Previous studies have shown multiple sequence alignments highlighting the conservation

395

of amino acids in EsxG and EsxH orthologs from several mycobacterial strains, but not from

396

Mab [13]. We re-performed multiple sequence alignments of the conserved amino acids in

397

EsxG (MAB 2229c, upper) and EsxH (MAB 2228c, lower) orthologs from Mab, Mtb H37Rv,

398

Mtb H37Ra, M. bovis, M. ulcerans, M. leprae, M. smegmatis, and M. marinum (Fig.6A).

399

Overall, the amino acid sequences of orthologs were well conserved.

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To evaluate the immunological relevance of both proteins, recombinant EsxGH (rEsxGH)

402

complex was purified. A previous study showed that the Mtb proteins CFP‐10 (Rv0287) and

403

ESAT‐6 (Rv0288), homologue proteins of EsxG and EsxH, respectively, form tight

404

heterodimeric complexes in the solution [30]. Consistent with the previous result, size-

405

exclusion chromatography analysis revealed that EsxG and EsxH proteins were co-eluted at

406

a fraction corresponding to the EsxGH complex with a molecular weight of ~20 kDa (Fig.6B).

407

The complex was confirmed by SDS-PAGE analysis (Fig.6C).

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Subsequently, BMDMs were stimulated in vitro with purified rEsxGH and then subjected

410

to endotoxin removal using polymyxin B-agarose. The cellular responses were analyzed and

411

compared with the response to Mab WT and ∆esx. As seen in Fig.6D, treatment of BMDMs

412

with rEsxGH protein alone did not induce the release of TNF-α or IL-6. Proinflammatory

413

cytokines were induced at a very low level of TNF-α and IL-6 from BMDMs infected with

414

∆esx, as seen in Fig.3. Interestingly, the production of TNF-α and IL-6 was synergistically

415

increased in ∆esx-infected BMDM when combined with rEsxGH protein in a dose-dependent

416

manner. These data suggest that rEsxGH from the ESX-3 region can rescue the function of

417

the ESX-3 region through in vitro complementation. Together, the EsxG and EsxH proteins

418

of the Mab ESX-3 region are associated with host inflammatory responses during the course

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of Mab infection.

420 421 422

4. Discussion In this study, we characterized the innate immune function of the Mab ESX-3 region by

424

constructing an ESX-3 (MAB 2224c-2234c)-deletional mutant strain of Mab (∆esx) in murine

425

bone marrow-derived macrophages (BMDMs) and in vivo. Compared to Mab wild type (Mab

426

ATCC 19977), ∆esx infection resulted in induction of less histopathology and inflammatory

427

mediator production in vivo. It also resulted in lower levels of inflammatory signaling and

428

cytokine production in macrophages. Additionally, intracellular bacterial survival was

429

significantly attenuated in ∆esx-infected conditions. In vitro complementation with

430

recombinant EsxG▪EsxH (rEsxGH) proteins, two major proteins encoded by the ESX-3

431

region in Mab [13], was also performed. This showed that restoration of inflammatory

432

cytokine production was as likely as with the wild-type strain. The present study reports

433

evidence showing an important role for Mab ESX-3 in the induction of host

434

immunopathological responses and increased mycobacterial growth during infection.

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Mab is one of the most frequent causes of lung disease, accounting for around 80% of

437

pulmonary infections caused by rapidly growing mycobacteria [3]. One of the most serious

438

clinical burdens in an emerging Mab infection is chemotherapy difficulties, due to the intrinsic

439

drug resistance of Mab to various antibiotics [31]. There is an urgent need for the

440

development of new vaccines and therapeutic strategies to control Mab infection in both

441

immunocompetent and immunocompromized patients [32,33]. To this end, a more

442

comprehensive understanding of the host–pathogen interaction should be preceded by the

443

identification and functional characterization of essential Mab genes. Our findings identify the

444

Mab ESX-3 region as important for intracellular bacterial survival and elicitation of excessive

445

inflammatory and pathological responses during Mab infection.

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ACCEPTED MANUSCRIPT 446

In more detail, deletion of the Mab ESX-3 region leads to attenuated growth and

448

persistence in both macrophages. In addition, ∆esx infected organs such as spleen and liver

449

rapidly underwent a progressive reduction of CFU in comparison with wild-type strain at 7

450

days after infection. Mycobacterial virulence can be measured by the ability of bacteria to

451

invade, grow, and persist not only in macrophages, but also in an in vivo rodent model.

452

Sweeney et al. previously showed that the IKE (Mycobacterium smegmatis strain with

453

deletion of the esx-3 region) strain could not elicit different IL-6 amounts with parental and

454

∆esx-1 Msmeg. Fredric et al. also reported that Esx-1 deficient (∆RD1) M. marinum

455

appeared to perturb the host immune response [34]. In more detail, ∆RD1 M. marinum alters

456

the immune response by decreasing TNFα and IL-6 production. The presence of Esx-1

457

promotes NFκB activation in macrophages in vitro, which could account for increased TNFα

458

and IL-6 seen with infections by WT M. marinum in vivo. In this study, we observed innate

459

immune responses that were similar to those seen in the host immune response of ∆RD1 M.

460

marinum. This is because the Mab ESX-3 cluster harbors esxG (Rv0287, ESAT-6 like

461

protein), which has high homology with M. marinum esxG (MMAR 0546; ESAT-6 like protein)

462

(74% identities and 80% positives). The generation of proinflammatory cytokines (TNF-α, IL-

463

6, IL-1β, and IL-12p40) is significantly decreased by the ∆esx strain in macrophages from

464

mice and humans. This is the first report that Mab ESX-3 is involved in exacerbated

465

inflammatory responses, as well as disease pathology and reduced bacillary control, during

466

infection. TNF-α is a key proinflammatory cytokine that mediates mycobacterial killing, host

467

defense, chemokine expression, and granuloma formation [35,36]. However, excessive

468

levels of TNF-α can lead to detrimental effects in the host. For example, excessive secretion

469

of TNF-α has been implicated in clinical worsening and tissue damage, whereas TNF-α

470

reduction has been correlated with decreased granuloma size and necrosis [37]. Thus, the

471

appropriate induction and control of this cytokine are thought to be key in host-directed

472

responses against tuberculosis [38]. Our report suggests that the Mab esx-3 region is

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ACCEPTED MANUSCRIPT 473

involved in serum cytokine production that subsequently influences virulence in a mouse

474

model of infection.

475

Inflammatory responses triggered by mycobacterial infection lead to the robust

477

activation of intracellular signaling cascades involving three subfamilies of MAPKs and NF-

478

κB activation [8,26]. We also found that macrophage inflammatory signaling (NF-κB and

479

MAPK) is markedly downregulated by ∆esx infection. Indeed, there have been many reports

480

of in vitro and in vivo immune-modulating activity of the ESAT-6 antigen of Mtb. The ESAT-6

481

antigen elicited vaccine-enhancing Th17 immune responses through TLR2/MyD88 signaling

482

[39]. In contrast, RD-1 region antigens played the key role of down-regulating the functions

483

of macrophages, potentially contributing to Mtb virulence [40]. Previous studies also showed

484

that disruption of the M. marinum PPE38 gene resulted in reduced levels of TNF-α and IL-6

485

secretion in infected macrophages [41]. BLAST analysis revealed that MAB_2230c within

486

the ESX-3 gene cluster shares intermediate homology (~35%) with M. marinum PPE38

487

(data not shown). Taken together, our data suggest that the ESX-3 region may contribute to

488

excessive inflammatory responses and signaling activation in host macrophages.

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Efficiently mounting host defenses against mycobacterial infection depends on both

491

restriction of bacterial replication and prevention of overwhelming inflammatory responses

492

[42]. The immunopathological responses to Mab are poorly characterized compared to the

493

widely studied Mtb infection. The name “Mab” originated from abscess formation during

494

infection [43]. Clinical manifestations of Mab infection are often related to abscess

495

formations that require surgical treatment and severe inflammatory responses, such as

496

ulcerative, abscess-forming lesions [44,45]. In this study, we report that Mab infection

497

resulted in markedly increased granulomatous infiltrate and inflammatory infiltrates in mouse

498

lungs. We also found that the infiltration of neutrophils and immune cells expressing COX-2

499

and iNOS was markedly increased in mouse tissues within 1 week of infection with the Mab

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ACCEPTED MANUSCRIPT WT strain. Notably, pathological responses found in Mab WT-infected lungs and other

501

tissues were significantly attenuated compared to those from ∆esx-infected mice. These

502

data indicate that the Mab ESX-3 region is important in Mab pathogenesis and virulence

503

during infection.

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504

In this study, we were unable to complement the Mab WT strain with a mutant strain

506

given the current genetic tools. As with the M. smegmatis IKE strain, we could not

507

complement with cosmid pYUB1336, which harbors an intact M. tuberculosis ESX-3 locus

508

(Rv0282–Rv0292) [20]. We speculate that we would have been unable to complement Esx-3

509

function in an M. smegmatis mutant even if it were expressed pYUB1336 in this species,

510

suggesting that esx-3 functions are species-specific. Other E. coli/ Mycobacterial expression

511

vectors using the hsp60 promoter fused with the ESX-3 region (MAB 2224c-2234c) were

512

also unable to complement, probably due to overexpression of a gene cluster with several

513

membrane proteins. To compensate for this in an alternative way, recombinant proteins

514

rEsxG and rEsxH were generated from esxH and esxH genes, which were expected to be

515

the main functional genes in the Mab ESX-3 region. The ∆esx phenotype was partly rescued

516

when ∆esx was co-incubated with rEsxG and rEsxH. Although the recombinant protein alone

517

was unable to induce proinflammatory cytokine release, ∆esx - rEsxG and rEsxH could elicit

518

a dose-dependent increase in TNF-α and IL-6 levels in macrophages to levels observed in

519

Mab WT strain-infected cells. Recent studies showed that Mtb type VII effector EsxH, in

520

complex with EsxG, is involved in impaired phagosomal maturation by disrupting the

521

endosomal sorting complex required for transport (ESCRT) function, restricting intracellular

522

bacterial growth [46]. Combined with our data, this suggests that at least two proteins, esxG

523

and esxH gene products, may contribute to the pathological responses induced by the Mab

524

ESX-3 region. Further studies are needed to clarify the distinct function of each gene located

525

within the ESX-3 region of the Mab genome.

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ACCEPTED MANUSCRIPT 526

In summary, we report a function of the Mab ESX-3 cluster with regard to virulence and

528

replication failure in a mouse model of infection. The absence of Mab ∆esx resulted in less

529

accumulation of neutrophils and inflammatory mediator release in vivo. Furthermore, Mab

530

ESX-3 plays an important role in excessive proinflammatory cytokine production through

531

modulation of MAPKs and NF-κB in macrophages. The EsxGH proteins of the Mab ESX-3

532

region contributed to a rescue of the attenuated inflammatory responses induced by the

533

∆esx strain. The immunopathological action of the Mab ESX-3 region may be associated

534

with Mab virulence and inflammatory responses during the course of Mab infection.

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Acknowledgements

We thank D. M. Shin and J. J. Kim for helpful discussion and reagents. We thank D.

538

Ray for critical reading of the manuscript. This work was supported by a grant of the Korea

539

Health Technology R&D Project through the Korea Health Industry Development Institute

540

(KHIDI), by the National Research Foundation of Korea (NRF) grant funded by the Korean

541

government (MSIP) (2011-0030049) at Hanyang University, and by the Basic Science

542

Research Program through the NRF funded by the Ministry of Education (NRF-

543

2014R1A6A1029617) .

545 546 547 548 549 550

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Conflict of interest

No conflict of interest

ACCEPTED MANUSCRIPT References

552

[1] Field SK, Cowie RL. Lung disease due to the more common nontuberculous

553

mycobacteria. Chest 2006; 129:1653-72.

554

[2] Brown-Elliott BA, Wallace RJ Jr. Clinical and taxonomic status of pathogenic

555

nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev

556

2002;15:716-46.

557

[3] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official

558

ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial

559

diseases. Am J Respir Crit Care Med 2007;175:367-416.

560

[4] Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: a new

561

antibiotic nightmare. J Antimicrob Chemother 2012;67:810-8.

562

[5] Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT, Engele M, Sieling PA, et al.

563

Induction of direct antimicrobial activity through mammalian toll-like receptors. Science

564

2001;291:1544-7.

565

[6] Yamashiro LH, Oliveira SC, Báfica A. Innate immune sensing of nucleic acids from

566

mycobacteria. Microbes Infect 2014;16:991-7.

567

[7] Stamm CE, Collins AC, Shiloh MU. Sensing of Mycobacterium tuberculosis and

568

consequences to both host and bacillus. Immunol Rev 2015;264:204-19.

569

[8] Basu J, Shin DM, Jo EK. Mycobacterial signaling through toll-like receptors. Front Cell

570

Infect Microbiol 2012;2:145.

571

[9] Killick KE, Ní Cheallaigh C, O'Farrelly C, Hokamp K, MacHugh DE, Harris J. Receptor-

572

mediated recognition of mycobacterial pathogens. Cell Microbiol 2013;15:1484-95.

573

[10] Hossain MM, Norazmi MN. Pattern recognition receptors and cytokines in

574

Mycobacterium

575

2013;2013:179174.

576

[11] Howard ST. Recent progress towards understanding genetic variation in the

577

Mycobacterium abscessus complex. Tuberculosis (Edinb) 2013;93:S15-20.

AC C

EP

TE D

M AN U

SC

RI PT

551

tuberculosis

infection--the

double-edged

sword?

Biomed

Res

Int

ACCEPTED MANUSCRIPT [12] Stanley SA, Raghavan S, Hwang WW, Cox JS. Acute infection and macrophage

579

subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl

580

Acad Sci USA 2003;100:13001-6.

581

[13] Ilghari D, Lightbody KL, Veverka V, Waters LC, Muskett FW, Renshaw PS, et al.

582

Solution structure of the Mycobacterium tuberculosis EsxG.EsxH complex: functional

583

implications and comparisons with other M. tuberculosis Esx family complexes. J Biol Chem

584

2011;286:29993-30002.

585

[14] Liu W, Peng Y, Yin Y, Zhou Z, Zhou W, Dai Y. The involvement of NADPH oxidase-

586

mediated ROS in cytokine secretion from macrophages induced by Mycobacterium

587

tuberculosis ESAT-6. Inflammation 2014;37:880-92.

588

[15] Lewis KN, Liao R, Guinn KM, Hickey MJ, Smith S, Behr MA, et al. Deletion of RD1 from

589

Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation. J Infect Dis

590

2003;187:117-23.

591

[16] Abdallah AM, Savage ND, van Zon M, Wilson L, Vandenbroucke-Grauls CM, van der

592

Wel NN, et al. The ESX-5 secretion system of Mycobacterium marinum modulates the

593

macrophage response. J Immunol 2008;181:7166-75.

594

[17] Choo SW, Wee WY, Ngeow YF, Mitchell W, Tan JL, Wong GJ, et al. Genomic

595

reconnaissance of clinical isolates of emerging human pathogen Mycobacterium abscessus

596

reveals high evolutionary potential. Sci Rep 2014;4:4061.

597

[18] Sassi M, Drancourt M. Genome analysis reveals three genomospecies in

598

Mycobacterium abscessus. BMC Genomics. 2014;15:359.

599

[19] Siegrist MS, Unnikrishnan M, McConnell MJ, Borowsky M, Cheng TY, Siddiqi N, et al.

600

Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc Natl Acad Sci

601

USA 2009;106:18792-7.

602

[20] Sweeney KA, Dao DN, Goldberg MF, Hsu T, Venkataswamy MM, Henao-Tamayo M, et

603

al. A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against

604

Mycobacterium tuberculosis. Nat Med 2011;17:1261-8.

AC C

EP

TE D

M AN U

SC

RI PT

578

ACCEPTED MANUSCRIPT [21] Serafini A, Pisu D, Palù G, Rodriguez GM, Manganelli R. The ESX-3 secretion system is

606

necessary for iron and zinc homeostasis in Mycobacterium tuberculosis. PLoS One.

607

2013;8:e78351.

608

[22] Yang CS, Shin DM, Kim KH, Lee ZW, Lee CH, Park SG, et al. NADPH oxidase 2

609

interaction with TLR2 is required for efficient innate immune responses to mycobacteria via

610

cathelicidin expression. J Immunol 2009;182:3696-705.

611

[23] Wang J, Li BX, Ge PP, Li J, Wang Q, Gao GF, Qiu XB, Liu CH. Mycobacterium

612

tuberculosis suppresses innate immunity by coopting the host ubiquitin system. Nat Immunol.

613

2015;16:237-45.

614

[24] Sheffield P, Garrard S, Derewenda Z. Overcoming expression and purification problems

615

of RhoGDI using a family of "parallel" expression vectors. Protein Expr Purif 1999;15:34-9.

616

[25] van Kessel JC, Marinelli LJ, Hatfull GF. Recombineering mycobacteria and their phages.

617

Nat Rev Microbiol 2008;6:851-7.

618

[26] Shin DM, Yang CS, Yuk JM, Lee JY, Kim KH, Shin SJ, et al. Mycobacterium abscessus

619

activates the macrophage innate immune response via a physical and functional interaction

620

between TLR2 and dectin-1. Cell Microbiol 2008;10:1608-21.

621

[27] Zhou B, He Y, Zhang X, Xu J, Luo Y, Wang Y, et al. Targeting mycobacterium protein

622

tyrosine phosphatase B for antituberculosis agents. Proc Natl Acad Sci USA 2010;107:4573-

623

8.

624

[28] Seto S, Tsujimura K, Koide Y. Coronin-1a inhibits autophagosome formation around

625

Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in

626

macrophages. Cell Microbiol 2012;14:710-27.

627

[29] Kim TS1, Kim YS, Yoo H, Park YK, Jo EK. Mycobacterium massiliense induces

628

inflammatory responses in macrophages through Toll-like receptor 2 and c-Jun N-terminal

629

kinase. J Clin Immunol 2014;34:212-23.

630

[30] Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G, Frenkiel TA, et al.

631

Structure and function of the complex formed by the tuberculosis virulence factors CFP-10

AC C

EP

TE D

M AN U

SC

RI PT

605

ACCEPTED MANUSCRIPT and ESAT-6. EMBO J 2005;24:2491-8.

633

[31] Lee MR, Sheng WH, Hung CC, Yu CJ, Lee LN, Hsueh PR. Mycobacterium abscessus

634

complex infections in humans. Emerg Infect Dis 2015;21:1638-46.

635

[32] Oh CT, Moon C, Park OK, Kwon SH, Jang J. Novel drug combination for

636

Mycobacterium abscessus disease therapy identified in a Drosophila infection model. J

637

Antimicrob Chemother 2014;69:1599-1607.

638

[33] Abdalla MY, Switzer BL, Goss CH, Aitken ML, Singh PK, Britigan BE. Gallium

639

compounds exhibit potential as new therapeutic agents against Mycobacterium abscessus.

640

Antimicrob Agents Chemother 2015;59: 4826-34.

641

[34] Carlsson F, Kim J, Dumitru C, Barck KH, Carano RA, Sun M, et al. Host-detrimental role

642

of Esx-1-mediated inflammasome activation in mycobacterial infection. PLoS Pathog.

643

2010;6:e1000895.

644

[35] Bermudez LE, Young LS. Tumor necrosis factor, alone or in combination with IL-2, but

645

not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex. J

646

Immunol 1988;140:3006-13.

647

[36] Flynn JL, Goldstein MM, Chan J, Triebold KJ, Pfeffer K, Lowenstein CJ, et al. Tumor

648

necrosis factor-alpha is required in the protective immune response against Mycobacterium

649

tuberculosis in mice. Immunity 1995;2:561-72.

650

[37] Bekker LG, Moreira AL, Bergtold A, Freeman S, Ryffel B, Kaplan G. Immunopathologic

651

effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent.

652

Infect Immun 2000;68:6954-61.

653

[38] Dorhoi A, Kaufmann SH. Tumor necrosis factor alpha in mycobacterial infection. Semin

654

Immunol 2014;26:203-209.

655

[39] Chatterjee S, Dwivedi VP, Singh Y, Siddiqui I, Sharma P, Van Kaer L, et al. Early

656

secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17

657

cell responses in a toll-like receptor-2-dependent manner. PLoS Pathog 2011;7:e1002378.

658

[40] Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A, et al. Direct

AC C

EP

TE D

M AN U

SC

RI PT

632

ACCEPTED MANUSCRIPT extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium

660

tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 2007;8:610-8.

661

[41] Dong D, Wang D, Li M, Wang H, Yu J, Wang C, et al. PPE38 modulates the innate

662

immune response and is required for Mycobacterium marinum virulence. Infect Immun

663

2012;80:43-54.

664

[42] Nandi B, Behar SM. Regulation of neutrophils by interferon-γ limits lung inflammation

665

during tuberculosis infection. J Exp Med. 2011;208:2251-62.

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[43] Moore M, Frerichs JB. An unusual acid-fast infection of the knee with subcutaneous,

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abscess-like lesions of the gluteal region; report of a case with a study of the organism,

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Mycobacterium abscessus, n. sp. J Invest Dermatol 1953;20:133-169.

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[44] Song JY, Sohn JW, Jeong HW, Cheong HJ, Kim WJ, Kim MJ. An outbreak of post-

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acupuncture cutaneous infection due to Mycobacterium abscessus. BMC Infect Dis 2006;6:6.

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[45] Johnson MM, Odell JA. Nontuberculous mycobacterial pulmonary infections. J Thorac

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Dis 2014;6:210-20.

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[46] Mehra A, Zahra A, Thompson V, Sirisaengtaksin N, Wells A, Porto M, et al.

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Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair

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trafficking. PLoS Pathog 2013;9:e100373.

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Figure legends

687

Fig.1. Mab esx-3 gene cluster is required for in vitro and in vivo growth.

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A. Mab WT and ∆esx were grown in enriched Middlebrook 7H9 broth supplemented with

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0.05% Tween and ADC and OD value was taken every 12 h at 600 nm. All data was

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repeated in triplicates.

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B. BMDMs were infected with different MOI (MOI of 1 or 10) of both either Mab WT or ∆esx,

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washed, and macrophage lysates plated for CFU counts on 7H10 agar.

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C. C57BL/6 mice were intravenously infected with 1×107 CFU of Mab WT and ∆esx strains.

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Bacillary loads were determined in spleens and livers of C57BL/6 (n = 5) infected with Mab

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WT and ∆esx at 1, 7, and 14 days post-infection. All data points are represented by log10

697

CFU values, and bars indicate standard errors.

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D. Comparison of histopathological findings from C57BL/6 mice were intratracheally infected

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with 5×105 CFU of Mab WT and ∆esx strains or PBST mock control. The figure depicts a

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representative photograph of the extent of pathological damage of mice infected with PBST

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mock control (n = 3), Mab WT (n = 5), and ∆esx (n = 5) at 7 days post-infection. Quantitative

702

scoring of histopathology is shown in the left lower panel. Data are presented as the mean ±

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SEM of four independent experiments. *P < 0.05, **P <0.01, ***P < 0.001. Scale bars, 100

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µm.

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Fig.2. Esx-3 disruption induces less pro-inflammatory responses in vivo.

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A. Blood serum levels of pro-inflammatory cytokines such as TNF-α (left) and IL-6 (right)

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were measured by ELISA analysis from C57BL/6 mice were intravenously infected with

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1×107 CFU of Mab WT and ∆esx strains (n = 9).

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B. Expression of COX-2 and iNOS, as well as neutrophil infiltration in lung tissues from

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C57BL/6 mice were intratracheally infected with 5×105 CFU of Mab WT (n = 5) and ∆esx

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strains (n = 5) or PBST mock control (n = 3) at 7 days post-infection. Representative results

ACCEPTED MANUSCRIPT of immunohistochemistric analysis of expression of COX-2 and iNOS, and neutrophil

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infiltration in lung tissues from mice infected with Mab WT or ∆esx (left). A brown color

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indicates positive staining for the indicted proteins. Scale bars, 100 µm (for COX2 and iNOS)

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or 50 µm (for Neutrophil).

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C. Quantitative scoring of histopathologic findings (for B) by percentage of positively strained

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cells. *P < 0.05, **P <0.01, ***P < 0.001.

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Fig.3. Proinflammatory cytokine generation in murine and human macrophages after

721

infection with Mab WT or ∆esx strain.

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A and B. BMDMs were infected with Mab WT or ∆esx (MOI = 5) for the indicated times. The

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mRNAs and supernatants were collected and subjected to qRT-PCR (for A) or ELISA

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analysis (for B) to measure mRNA and protein expression of TNF-α, IL-6, IL-1β, and IL-

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12p40.

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C and D. Human MDMs were infected with Mab WT or ∆esx (MOI = 5) for the indicated

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times. The mRNAs and supernatants were collected and subjected to qRT-PCR (for C) or

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ELISA analysis (for D) to measure mRNA and protein expression of TNF-α and IL-6. Data

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are presented as the mean ± SEM of four independent experiments. *P < 0.05, **P <0.01,

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***P < 0.001.

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Fig.4. Comparison of MAPK activation in BMDMs infected with Mab WT or ∆esx.

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A and B. Immunoblot analysis of whole-cell lysates from BMDMs infected with Mab WT or

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∆esx (MOI = 5) for the indicated times (0-480 min) using antibodies against phosphorylated

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forms of ERK, p38, and JNK. β-actin served as loading control. WB images representative of

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three experiments are shown in panel A. Densitomety values for phospho-ERK, p-p38, and

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p-JNK were normalized to β-actin (for B). Data are presented as the mean ± SEM of four

738

independent experiments. **P <0.01, ***P < 0.001.

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C. Immunoblot analyses of BMDMs infected with Mab WT or ∆esx (MOI = 1, 5 or 10; for 30

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min) with antibodies raised to phosphorylated forms of ERK, p38, and JNK. β-actin served

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as loading control. WB images representative of three experiments are shown.

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Fig.5. Comparison of NF-κB signaling in BMDMs infected with Mab WT or ∆esx.

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A and B. BMDMs were infected with Mab WT or ∆esx (MOI = 1, 5 or 10) for 30 min, followed

745

by immunostaining with anti-NF-κB p65 Ab, anti-rabbit-Alexa Fluor 488 (green), and DAPI to

746

visualize nuclei (blue). Representative immunofluorescence images (for A) and average

747

mean fluorescence intensity of cells exhibiting NF-κB nuclear translocation (for B) is shown.

748

C. Analysis of NF-κB p65 luciferase activity. BMDMs were transduced with adenovirus

749

carrying NF-κB luciferase reporter constructs for 36 h and infected with Mab WT or ∆esx

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(MOI = 1, 5 or 10), or stimulated with LPS (100 ng/ml) for 6 h.

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D. Immunoblot analysis of whole-cell lysates from BMDMs stimulated with Mab WT or ∆esx

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(MOI = 5) for the indicated times (0-480 min) using antibodies against IκB-α. β-actin served

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as a loading control. Data are presented as the mean ± SEM of four independent

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experiments. *P < 0.05, ***P < 0.001.

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Fig.6. Purification and immunological characterization of EsxG▪EsxH in BMDMs during Mab

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infection.

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A. Multiple sequence alignments of conserved amino acids in EsxG (MAB_2229c, upper)

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and EsxH (MAB_2228c, lower) ortholog from various mycobacterial strains (Mab, Mtb

760

H37Rv, Mtb H37Ra, M. bovis, M. ulcerans, M. leprae, M. smegmatis, and M. marinum).

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B. Size-exclusion chromatography of rEsxGH complex. Mixture of purified EsxG and EsxH

762

proteins was injected onto Superdex 75 10/300 GL column. Elution peak corresponding to

763

EsxGH complex with a molecular weight of ~20 kDa is indicated.

764

C. SDS-PAGE analysis of eluted EsxG and EsxH proteins. Proteins were visualized by

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Coomassie blue staining. M, mixture of purified EsxG and EsxH proteins. His-tagged EsxG

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(upper arrow) and tag-free EsxH (lower arrow) are indicated, respectively.

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D. BMDMs were treated with purified rEsxGH (5, 10 or 20 µg/ml, for 1 h), followed by

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infection with ∆esx. They were then subjected to ELISA analysis to detect levels of TNF-α

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and IL-6 at 18 h. Data are presented as the mean ± SEM of four independent experiments.

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***P < 0.001.

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Supplementary figure 1. Construction of Mab mutants using recombineering system.

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A and B. Schematic representation of genetic organization of esx-3 gene cluster in Mab WT

774

genome (for A) and gene disruption by zeocin cassette (for B). Position of oligonucleotides

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(primer-F and primer-R) used for PCR validation and expected sizes of resulting amplicons

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are indicated. C and D. ∆esx mutants were confirmed with primers shown (for C) and

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individual gene deletions were analyzed with different primers (for D).

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Figure 1.

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Figure 6.