PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and trigger cell death

Accepted Manuscript PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and trigger cell death Xuan Peng, Tao Luo, Xiaoqian Zhai, Chunxi Zhang, Jing Suo, Pengjiao Ma, Chuhan Wang, Lang Bao PII:

S0882-4010(18)30092-5

DOI:

https://doi.org/10.1016/j.micpath.2018.10.031

Reference:

YMPAT 3225

To appear in:

Microbial Pathogenesis

Received Date: 4 February 2018 Revised Date:

9 August 2018

Accepted Date: 22 October 2018

Please cite this article as: Peng X, Luo T, Zhai X, Zhang C, Suo J, Ma P, Wang C, Bao L, PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and trigger cell death, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.10.031. 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.

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ACCEPTED MANUSCRIPT PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and

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trigger cell death

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Xuan Penga, Tao Luoa, Xiaoqian Zhaia, Chunxi Zhanga, Jing Suoa, Pengjiao Maa,

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Chuhan Wanga, Lang Baoa*

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Affiliations: a Laboratory of Infection and Immunity, West China School of Basic

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Sciences & Forensic Medicine, Sichuan University, Chengdu, China.

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Corresponding author: Prof. Lang Bao. Laboratory of Infection and Immunity, West

China School of Basic Sciences & Forensic Medicine, Sichuan University, Chengdu,

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610041, China. E-mail: [email protected]; Tel: +86-28-85501237

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Abstract

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Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a

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serious global health problem. The PE/PPE family, featuring unique sequences,

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structures and expression in Mtb, is reported to interfere with the macrophage

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response to the pathogen and facilitate its infection. PPE11 (Rv0453) existed in

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pathogenic mycobacteria and was persistently expressed in the infected guinea pig

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lungs. However, the role it played in the pathogenesis remains unclear. Here, to

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investigate the interaction and potential mechanism of PPE11 between pathogens and

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hosts, we heterologously expressed PPE11 in non-pathogenic, rapidly growing

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Mycobacterium smegmatis strains. We found that the overexpression of the cell

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wall-associated protein, PPE11, can improve the viability of bacteria in the presence

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of lysozyme, hydrogen peroxide and acid stress. Expression of PPE11 enhanced the

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early survival of M. smegmatis in macrophages and sustained a higher bacterial load

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in mouse tissues that showed exacerbated organ pathology. Macrophages infected

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with recombinant M. smegmatis produced significantly greater amounts of interleukin

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（IL）-1β, IL-6, tumour necrosis factor (TNF)-α and an early decrease in IL-10 along

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with higher levels of host cell death. Similar cytokines changes were observed in the

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sera of infected mice. Accordingly, PPE11 protein causes histopathological changes

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by disrupting the dynamic balance of the inflammatory factors and promoting

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host-cell death, indicating a potential role in the virulence of Mtb.

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ACCEPTED MANUSCRIPT Keywords: Mycobacterium tuberculosis; PE/PPE; PPE11; Mycobacterium

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smegmatis; pathology; Virulence

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1. Introduction:

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Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection remains a

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serious global health threat. According to the World Health Organization's annual

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report, there is an estimated number of 10.4 million people that have fallen ill with

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TB worldwide in 2016 [1]. The continuous rise of human immunodeficiency virus

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(HIV) co-infection and drug resistance further increases the mortality rate of TB [2-5].

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As an intracellular pathogen, Mtb can modulate and elude the host immune responses,

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which is beneficial to its intracellular survival for a prolonged period [6]. Therefore, a

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better understanding the molecular mechanisms of the interactions between Mtb and

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the host immune system is critical for the development of effective vaccines and

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anti-TB drugs.

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The genomic sequence of Mtb encodes approximately 4,000 genes, of which

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approximately 40% are predicted as putative proteins with unknown functions [7].

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According to the genomic annotation, 100 and 69 genes have been grouped as the

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proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein family,

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accounting for almost 10% of the coding capacity of the Mtb genome [7, 8]. The PPE

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subfamily has a conserved N-terminal domain of 180 amino acids characterized by

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the Pro (P) -Pro (P) -Glu (E) sequence, namely PPE domain. PPE proteins of Mtb

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were further divided into four subtypes according to their C-terminal domains,

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including the PPE_SVP, PPE_PPW, PPE_MPTR and special type PPE subfamilies.

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ESX-related genes encoding type VII secretion systems that is associated with Mtb

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virulence, as such the PPE protein were thought to have contributed to the

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pathogenesis of Mtb [8]. Studies have shown that some PPE proteins are secreted to

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the bacterial surface and interact with other proteins and host immune system

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components [9-11]. Recent data suggested that PPE proteins have a role in immune

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escape during Mtb infection [12]. Many PE/PPE proteins interact with Toll-like

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receptor 2 (TLR-2) and induce the release of cytokines by activating macrophages

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and dendritic cells, promoting host cell death [13-15]. Some PPE proteins, such as the

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PE25/PPE41 protein complex, augment macrophage necrosis [16]. In addition, PPE

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proteins could interfere with macrophage’s secretion of cytokines to regulate the

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dynamic balance of inflammatory responses [17, 18]. Therefore, clarifying the

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interactions between PPE proteins and host immunity is essential for understanding

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the pathogenicity of Mtb.

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ACCEPTED MANUSCRIPT According to the homologous analysis, the ppe genes may have evolved from

The immune potential of Mtb PPE proteins have been increasingly explored in the

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last decade, however, many of them are still rarely studied, among which the protein

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members of the PPW subfamily belonging to the PPE family have attracted the

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attention of scholars. The C-terminus of the PPE-PPW subfamily consists of 44

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amino acid residues, including the highly-protected Gly-Phe-X-Gly-Thr and

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Pro-X-X-Pro-X-X-Trp sequence motifs [19]. In recent years, scholars have paid

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attention to the function of PPE2 protein belonging to the PPE_PPW subfamily.

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translocate to the host nucleus and inhibit the production of nitric oxide [20, 21].

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However, PPE11 belonging to the same subfamily is still rarely studied. PPE11 is

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encoded by Rv0453 and its expression could be continuously detected in the lungs of

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guinea pigs 30 days post infection [22]. PPE11 is regulated by Lsr2, which is a

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metallogenic nucleotide-related transcriptional inhibitor and is associated with

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adapting to altered oxygen levels, to promote mycobacterium adaptation to adverse

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conditions [7, 23]. However, the interaction between PPE11 and host immune factors

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remains unclear. In the current study, we constructed a recombinant M. smegmatis

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strain expressing Mtb PPE11, and subsequently assessed the effect of recombinant

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strains on bacterial survival in different stress conditions, as well as in macrophages

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and mice. Our results shows that the expression of PPE11 in M. smegmatis can cause

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pathological changes in mice by interfering with the secretion of inflammatory

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cytokines and promoting host cell death. In addition, our data indicates that the

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expression of PPE11 may help M. smegmatis to invade host cells and establish a

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persistent infection and may play an important role in the virulence of Mtb.

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ACCEPTED MANUSCRIPT Studies have shown that PPE2 can not only induce strong B cell responses, but also

2. Material and methods

2.1 Bacterial strains, cell lines and animals

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Escherichia coli (E. coli) BL21 (Vazyme, China) strains for DNA cloning were grown 6

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smegmatis mc2155 and recombinant M. smegmatis were grown in Middlebrook 7H9

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broth (M7H9, BD Difco, USA) supplemented with 10% oleic acid–albumin–

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dextrose–catalase (OADC), 0.5% (v/v) glycerol and 0.05% (v/v) Tween-80

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(Sigma-Aldrish, USA), or on Middlebrook 7H10 agar (M7H10, BD Difco, USA)

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supplemented with 10% OADC. Antibiotics were added according to need. THP-1

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cells were cultured in RPMI1640 medium supplemented with 10% foetal bovine

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serum (FBS) and 100 U/ml penicillin, 100 U/ml streptomycin (HyClone, USA) at

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37°C with an atmosphere of 5% CO2. BALB/c mice were purchased from

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Chengdu-Dossy (Chengdu, China). All mice were housed in a specific pathogen-free

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facility using standard humane animal husbandry protocols. Animal experiments were

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conducted in accordance to Laboratory Animal Care and approved by the Ethical

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Review Committee of Sichuan University.

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ACCEPTED MANUSCRIPT at 37◦C using Luria–Bertani (LB) broth supplemented with 50 µg/ml kanamycin. M.

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2.2 Construction and expression of recombinant M. smegmatis

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The ppe11 (Rv0453) gene was PCR-amplified using ppe11-F (Table 1), which

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contained a BamHI site (underlined), and reverse primer ppe11-R (Table 1)

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containing a HindIII site (underlined) and six His codons (lower-case letters) using

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Mtb H37Rv genomic DNA as a template. The resultant PCR product containing six

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consecutive His codons was ligated into the pMV261 shuttle vector, generating

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pMV261-6×hisPPE11.

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The

recombinant

plasmid

or

empty pMV261

was 7

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Recombinant M. smegmatis expressing 6× His-tagged ppe11 (Ms_PPE11) and M.

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smegmatis harbouring empty pMV261 alone (Ms_Vec) was selected on M7H10 agar

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containing 25 µg/ml kanamycin.

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ACCEPTED MANUSCRIPT electroporated into M. smegmatis mc2155 according to standard procedures.

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To detect the transcription of ppe11, recombinant M. smegmatis strains were cultured

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in 100 ml of M7H9 liquid medium in the presence of 25 µg/ml kanamycin until the

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OD600 was 0.6-1.0. Total RNA isolation was performed using Trizol reagent

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(Invitrogen, USA) according to the manufacturer’s instructions. All RNA samples

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were treated with RNase-free DNase I (Invitrogen, USA). The concentration of the

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total RNA was measured using a SmartSpec Plus spectrophotometer and a standard

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colorimetric cup (Bio-Rad, Laboratories, USA) of 1 cm path length according to the

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manufacturer’s instructions. Single-stranded cDNA was synthesized using 200 ng of

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total RNA as template and random hexamer primers according to the specification of

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the PrimeScriptTM RT kit (Kakara, Japan). Each cDNA sample was subjected to PCR

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amplification using the following primers: ppe11-F (i) and ppe11-R (i) (Table 1) for

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the ppe11 gene with a product size of 348 bp; And aph-F and aph-R (Table 1)

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aminoglycoside phosphotransferase (aph) genes, with a product size of 470 bp.

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To detect the PPE11 protein level, Ms_PPE11 and Ms_Vec were cultured. Protein

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samples were prepared as described previously. The PPE11 protein was separated by

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sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8

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China) and HRP conjugated goat anti-mouse IgG antibody (NeoBioscience, China).

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Ms_Vec was used as a negative control. Hsp65 is a widely used internal reference

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protein by immunoblotting of mycobacteria [24] and was detected using an antibody

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to Hsp65 (BDI578, Abcam) at a 1:1,000 dilution. ECL luminescent reagent (Sangon

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Biotech, China) was used to detect the signal according to the manufacturer's

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instructions.

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2.3 Subcellular localization of the PPE11 protein

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ACCEPTED MANUSCRIPT further detected by Western blot analysis with mouse anti-His tag antibody (Tiangen,

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The subcellular localization of the PPE11 protein was determined using the

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previously described method [25]. Briefly, bacteria cells were lysed by sonication for

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30 minutes. Intact cells and cell debris was removed by centrifugation at 3,000 g for 5

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minutes. The supernatant was centrifuged at 27,000 g for 40 minutes at 4℃. The

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supernatant was treated as a cell membrane and a cytoplasmic fraction, and the

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precipitate of the centrifugation step was regarded as a cell wall fraction. PPE11

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protein expression from each fraction was confirmed by Western blotting using a

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monoclonal antibody against His (dilution, 1: 1000; Tiangen, China). Ms_Vec was

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used as a negative control. The primary native GroEL2 was detected as a cytoplasmic

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control [26]. Natural GroEL2 was detected using anti-HSPDE mouse antibody

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(dilution, 1: 1000; Sangon, China) followed by HRP-conjugated goat anti-mouse IgG

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(Sangon, China). The band is displayed by enhanced chemiluminescence.

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2.4 In vitro infection with recombinant M. smegmatis.

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THP-1 cells were suspended in complete RPMI and seeded into 12-well plates at 5 ×

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105 cells/well. For infection, the suspended THP-1 cells were transformed into

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anchorage-dependent macrophages after 48 hours (h) treatment with 100 µg/ml

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phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrish, USA) at 37°C in 5% CO2.

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Exponentially growing bacteria cultured in the presence of 25 ug/mL kanamycin

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were pelleted and suspended in RPMI-1640 medium in a single-cell suspension.

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Ms_Vec and Ms_PPE11 were added to the macrophages kept with complete RPMI

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without antibiotics at a multiplicity of infection (MOI) of 10:1, respectively.

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Phagocytosis was permitted during a 4 h incubation at 37°C in 5% CO2, after which

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the macrophage cells were washed thoroughly with phosphate-buffered saline (PBS)

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to remove bacteria further. RPMI containing 100 ug/ml gentamicin was added to the

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infected macrophages after 4 h of infection to kill the remaining extracellular bacteria.

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Each sample has three replicates in this experiment.

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2.5 Measurement of the growth of recombinant M. smegmatis in vitro and in vivo

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To monitor the growth tendency of the recombinant M. smegmatis strains, the wild

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type M. smegmatis, Ms_Vec and Ms_PPE11, were shaken at 37°C in M7H9 liquid

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medium in a starting absorbance (OD600) of 0.02. The OD600 of the culture was 10

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ACCEPTED MANUSCRIPT continuously determined at a 3 h interval over a 48 h growth period, and average

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values were used to plot the growth curves. Each analysis was performed in triplicate.

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For the determination of bacterial survival within macrophages, M. smegmatis and

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cells were cultured as previously described [27]. The infected macrophages were

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washed by PBS three times and dissolved using 0.5% Triton-X-100 after 4, 24, 48

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and 72 h of infection. The cell lysates were serially diluted and plated onto M7H10

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agar supplemented with the appropriate antibiotic to determine the colony forming

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units (CFU).

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2.6 Measurement of sensitivity to lysozyme, hydrogen peroxide, low pH and nitric

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oxide

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To investigate the tolerance of recombinant M. smegmatis under different stress

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conditions, bacterial cultures were centrifuged and washed with M7H9 medium

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containing 0.05% Tween 80, and bacterial suspensions were adjusted to an

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absorbance at 600 nm of 0.5. To test the susceptibility of M. smegmatis strains to

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lysozyme, bacteria were treated with lysozyme (Sigma) (2500 µg/ml) for 1 h or 2 h at

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37°C. To measure the sensitivity towards low pH, cells were harvested by

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centrifuging and were washed with M7H9 (pH 3 or 5). Then, they were re-suspended

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at an OD600 of 0.5 in 5 ml M7H9 (pH 3 or 5) for 3, 6 or 9 h. To measure the

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sensitivity to hydrogen peroxide, the bacteria were incubated with 5 mM hydrogen

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acid sodium nitrite, the bacteria were incubated with 5 or 10 mM sodium nitrite

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(NaNO2) at pH 5.4 for 2, or 4 h at 37°C, and the CFUs for each stress were

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determined by plating serial dilutions onto M7H10 agar plates.

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ACCEPTED MANUSCRIPT peroxide (Merck, USA) for 1 h or 2 h at 37°C. Furthermore, to test susceptibility to

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2.7 Assay for lactate dehydrogenase release from macrophage

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Culture supernatants were harvested after the infection of differentiated THP-1

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macrophages with Ms_Vec or Ms_PPE11 for 6, 24, 48 or 72 h. Lactate

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dehydrogenase (LDH) activity in culture supernatants were assayed with the

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CytoTox96 Non-radioactive Cytotoxicity Assay Kit (Promega, USA) according to the

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manufacturer's instructions. The percentage of LDH release was calculated as follows:

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percentage release = 100×(experimental LDH release-spontaneous LDH release)/

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(maximal LDH release-spontaneous LDH release). The value of maximal LDH

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release was obtained from the complete lysis of uninfected THP-1 macrophages, and

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the spontaneous LDH release was measured with supernatants of uninfected cells.

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Each sample set has three repeats in this experiment.

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2.8 ELISA and Quantitative real-time PCR quantification of the expression of

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cytokines

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macrophages with Ms_Vec or Ms_PPE11 at 6, 24, 48, and 72 h intervals.

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Concentrations of TNF-α, IL-6, IL-1β, IL-4, IL-12p70 and IL-10 in the culture

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supernatants were determined with ELISA kits (eBioscience, USA) according to the

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manufacturer's instructions.

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ACCEPTED MANUSCRIPT Culture supernatants were harvested after infection of the differentiated THP-1

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PMA-differentiated THP-1 cells were infected with Ms_Vec and Ms_PPE11 at an

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MOI of 10. After 6 and 24 h infection, the total RNA was extracted from the infected

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cells using Trizol reagent (Invitrogen), according to the manufacturer’s

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recommendations. cDNA synthesis was performed using the PrimeScriptTM RT kit

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(Takara, Japan). Quantitative real-time RT-PCR reactions were performed using the

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Roche LightCycler 480II detection system using SYBR Premix Ex Taq Ⅱ (Takara,

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Japan). Relative mRNA levels were calculated after normalizing to β-actin. The

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primers are listed in Table 1.

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2.9 Infection of Balb/c mice with recombinant M. smegmatis strains

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Four to six-week-old female Balb/c mice (Chengdu-Dossy) were routinely fed for 2

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weeks prior to injection. Then, mice (6 mice per group) were injected with 5 × 107

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CFU of Ms_Vec or Ms_PPE11 by tail vein injection and the same dose of PBS was

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injected as a control. All strains were dispersed in PBS-T with no caking as required.

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The inoculum was cultured on an M7H10 plate containing kanamycin to determine 13

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3, 6 and 9 days), mice were sacrificed using the method described above. Briefly, the

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lung and spleen were aseptically removed and homogenized in sterile PBS-T. The

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mouse tissue homogenate was diluted in PBS-T and 100 µl aliquots from each

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dilution were plated on a M7H10 agar plate containing 10% OADC and 25 µg/ml

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kanamycin. The plates were incubated at 37°C and the CFU was used to determine

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the survival of the recombinant M. smegmatis strain in mouse organs after 3 days. To

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determine the serum cytokines in infected Balb /c mice, blood was collected from the

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mouse eyes on days 3, 6, 9 and 12 after infection and the serum was collected at

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2,500 g for 5 minutes [28]. Six mice were sacrificed at each time point. The levels of

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the serum cytokines TNF-α, IL-6, IL-1β, IL-10 and IL-4 were determined using an

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ELISA kit (eBioscience, USA) according to the manufacturer's instructions.

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ACCEPTED MANUSCRIPT the intravenous administration of an equal number of bacteria. At each time point (1,

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2.10 Histological analysis

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Mice injected with a recombinant strain through the tail vein were euthanized on the

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seventh day. The lung, liver and spleen of the mice were removed aseptically and

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fixed in 10% formalin. The tissue was embedded in paraffin and cut into a 4 µm

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section, mounted on a glass slide, dewaxed, and then stained with haematoxylin and

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eosin (H&E). Histopathological sections were examined by a clinical pathologist

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using

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photomicrographs were taken using an Olympus DP72 CCD camera attached to a

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microscope.

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2.11 Statistical analysis

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GraphPad Prism 6 software was used for statistical analysis. Data were expressed as

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the mean ± SD and Student’s t test was used to determine significant differences

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between the groups. P < 0.05 was considered to be significant.

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3. Results:

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3.1 The expression of PPE11 have no effect on the growth of recombinant M.

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smegmatis in vitro

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In this study, we constructed two recombinant strains of M. smegmatis: the strain

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Ms_PPE11 expressing the 6×His-labeled PPE11 protein and the strain Ms_Vec

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carrying the empty pMV261 vector. The introduction and transcription of PPE11 in

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Ms_PPE11 were validated by genomic PCR and reverse transcription PCR (RT-PCR)

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(Fig. 1A-B). In addition, a protein band of 55kDa representing PPE11 was detected

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from the Ms_PPE11 but absent in the Ms_Vec strain (Fig. 1C). To test the effect of

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PPE11 on the growth of recombinant strains, the growth rates of Ms_Vec and

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Ms_PPE11 were analysed in enriched M7H9 broth. No difference was detected

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ACCEPTED MANUSCRIPT between Ms_Vec and Ms_PPE11 (Fig. 1D), demonstrating that the expression of

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PPE11 does not affect the growth of M. smegmatis under aerobic conditions in vitro.

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3.2 PPE11 is associated with the cell wall

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To determine the localization of PPE11, Ms_Vec and Ms_PPE11 cultures were

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subjected to cell fractionation, followed by Western blot analysis with anti-His

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antibodies. As shown in Fig. 2, the majority of the PPE11 proteins were present in the

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cell wall fraction, suggesting that PPE11 is a cell wall-associated protein. As expected,

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the cytoplasmic heat-shock protein GroEL2 was detected only in the cytoplasm.

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3.3 Ms_PPE11 enhances the ability of recombinant bacteria to resist in vitro stress

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and early intercellular survival

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Macrophages are the primary cells infected by Mtb. Establishment of infection

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requires the bacteria to overcome multiple stress conditions, such as hypoxia,

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oxidative and acidic stress. To test whether PPE11 could help the bacteria to resistant

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these stresses, we compared the survival of Ms_PPE11 and Ms_Vec under lysozyme,

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hydrogen peroxide (H2O2), acidic and active nitrogen intermediates (RNI) conditions

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that mimic stresses in macrophages. We found Ms_PPE11 was more resistant to

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lysozyme and hydrogen peroxide (H2O2) than Ms_Vec at each experimental time

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point (Fig. 3A, B). In addition, Ms_PPE11 showed higher survival rate than Ms_Vec

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Finally, as demonstrated in Fig. 3D, no difference was found between Ms_PPE11 and

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Ms_Vec under RNI conditions. Taken together, these data suggest that PPE11 may

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have an effect on maintaining the elastic cell wall structure, which may help the

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non-pathogenic M. smegmatis to gain a longer persistence in macrophages. To

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investigate whether PPE11 could affect the survival of M. smegmatis in macrophages,

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we compared the intracellular viability of Ms_PPE11 and Ms_Vec in THP-1 cells

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after different time points of infection. As showed in Fig. 3E, Ms_PPE11 showed a

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higher bacterial presence compared to Ms_Vec at 24 h post infection. However, no

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difference was observed at 48 and 72 h post infection post infection. These results

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indicate that PPE11 may contribute to the survival of M. smegmatis in macrophages

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at an early stage after infection.

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3.4 PPE11 promotes the death of macrophage during infection

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ACCEPTED MANUSCRIPT at 9 h of acid stress treatment of pH 5 and at all time points of treatments of pH 3.

Mtb may manipulate host cell death to cause disease. Therefore, we examined the

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effects of PPE11 on inducing cell death by infecting THP-1 macrophages with

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recombinant M. smegmatis. As demonstrated in Fig. 4, there was no significant

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difference in LDH release between Ms_PPE11 and Ms_Vec at 6 h post infection.

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However, at 24, 48 and 72 h post infection, the macrophages infected by Ms_PPE11

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showed a significant increase in the LDH release. These results indicate that the

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expression of PPE11 by M. smegmatis triggered the death of macrophage.

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3.5 PPE11 selectively regulates the production of inflammatory cytokines by

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macrophages after M. smegmatis infection

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Inhibition of innate immune responses is critical for Mtb to establish successful

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infection. Therefore, we investigated whether Ms_PPE11 could affect secretion of

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innate-immune associated cytokines after infecting macrophages. Analysis of the

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cytokine levels in the supernatants by ELISA showed that the levels of the

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pro-inflammatory cytokine TNF-α were significantly increased from the

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macrophages infected with Ms_PPE11 at 6, 24, 48, and 72 h after infection (Fig. 5A).

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Concurrently, the levels of another pro-inflammatory cytokines, IL-6 and IL-1β, were

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significantly increased compared to the macrophages infected with Ms_PPE11 at 24,

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48, and 72 h after infection (Fig. 5B-C). There was no significant difference between

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IL-6 and IL-1β levels at 6 h after infection. In terms of anti-inflammatory, no

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difference were found in levels of IL-10 at 6 h post infection (Fig. 5D). Furthermore,

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no difference was observed in the levels of IL-4 and IL-12p70 between the two

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groups (data not show). Real-time quantitative PCR was used to detect the expression

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of TNF-α, IL-6, IL-1β and IL-10 at 6 h and 24 h after infection. The expression of

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TNF-α, IL-6 and IL-1β in THP-1 cells was upregulated by PPE11-expressing M.

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smegmatis infection (Fig. 5E-G). In addition, the expression of Ms_PPE11

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down-regulated the IL-10 mRNA levels at 6 h post-infection (Fig. 5H). Taken

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together, our data suggest that PPE11 regulates the innate immune response of

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inhibiting the production of anti-inflammatory cytokines. This shift in the balance

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between pro-inflammation and anti-inflammation during infection may lead to the

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resulting inflammation of the infection.

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ACCEPTED MANUSCRIPT macrophages by promoting the production of pro-inflammatory cytokines and

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3.6 Expression of PPE11 confers a survival advantage to M. smegmatis in a mouse

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model of infection.

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Several PE/PPE proteins are responsible for Mtb virulence [29, 30]. We next assessed

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the virulence property of PPE11 in vivo using an animal infection model. Ms_Vec and

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Ms_PPE11 were both dispersed into a single cell suspension and plated to give equal

378

amounts of bacteria in mice (Fig. 6A, Fig. S1). We found that Ms_PPE11 persisted

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significantly longer in all organs than Ms_Vec at almost all time points (Fig. 6B-D).

380

The mean CFU counts were significantly reduced in the lungs of Ms_Vec infected

381

mice compared to mice infected with the Ms_PPE11 strain at 3-9 days (Fig. 6B).

382

Similar observations were found in the livers (Fig. 6C) and in the spleens (Fig. 6D).

383

In addition, when the spleens were examined, we found that the size of the spleens

384

infected with the Ms_PPE11 strain were significantly larger than the Ms_Vec strain

385

infected (Fig. S2). These results indicate that the expression of PPE11 protein in

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non-pathogenic mycobacteria leads to a longer survival of the bacilli in the host cells,

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suggesting that PPE11 may play an important role in the persistence of mycobacteria.

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associated with the histopathological change of the organs [29]. Therefore, we

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assessed the histology of the lung, liver and spleen tissue of the mice infected with

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recombinant strains by histopathological examination of haematoxylin and eosin (H

393

& E) stained sections. We observed that Ms_PPE11-infected mice showed more

394

significant tissue damage than Ms_Vec infected mice (Fig. 7). On day 7 post infection,

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compared with the lungs of the mice infected with Ms_Vec, Ms_PPE11-infected mice

396

were observed to undergo alveolar wall epithelial cells necrosis, alveolar cavity

397

disappearance, and increased lymphocyte and phagocytic infiltration (Fig. 7A).

398

Similar pathological changes were observed in the liver of Ms_PPE11-infected mice.

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Ms_PPE11-infected mice showed severe damage in the liver, and we observed a great

400

deal of dot necrosis and infiltration of inflammatory cells (Fig. 7B). Meanwhile,

401

Ms_Vec infected mice liver sections showed slight inflammatory cell infiltration and

402

scattered necrotic liver cells on day 7 post infection. Histopathological analysis of the

403

spleen showed that there was extensive erythrocyte and inflammatory cell infiltration

404

in Ms_PPE11-infected mice on day 7 after infection (Fig. 7C). This is associated with

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a higher CFU count in the spleen tissue, which may be the cause of the enlarged

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spleen.

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ACCEPTED MANUSCRIPT Studies have shown that the burden of mycobacteria in mouse organs is usually

3.7 PPE11 regulates circulatory cytokine secretion in sera from infected mice

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We have observed that the expression of PPE11 can regulate the production of 20

412

infected mice, we examined the levels of inflammatory cytokines in the sera of mice

413

infected with recombinant strains 3, 6, 9 and 12 days after infection. Similar to our in

414

vitro data, we found that the mice infected with Ms_PPE11 had a relatively high level

415

of TNF-α and IL-1β at 6-9 days after infection compared with Ms_Vec infected mice

416

(Fig. 8A, C). At the same time, Ms_PPE11 increased the secretion of IL-6 cytokines

417

at 3 days after infection compared with Ms_Vec, but there was no difference in the

418

IL-6 levels at 6-12 days of infection (Fig. 8B). In addition, there was no difference in

419

the levels of IL-4 during infection (Fig. 8D). While the level of IL-10 cannot be

420

detected in the group. These results demonstrated that the expression of PPE11 by M.

421

smegmatis can induce pro-inflammatory cytokine production after infection. These

422

data may also suggest that the immunopathological changes in mouse tissues resulted

423

from PPE11-expressing M. smegmatis infection, which were related to the release of

424

cytokines in infected mice.

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4. Discussion:

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ACCEPTED MANUSCRIPT inflammatory cytokines in vitro (Fig. 5). To investigate the effect of PPE11 in

428

The ability of bacteria to adapt to the malignant environment and actively manipulate

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host signaling pathways determines the survival and persistence of Mtb in

430

macrophages. Many studies show that PPE proteins are involved in such process [31,

431

32].In the present study, to investigate the interaction of PPE11 with the host and its

432

possible biological functions, we heterologously expressed the PPE11 protein in the 21

434

PPE11 in M. smegmatis enhanced the early survival of recombinant bacteria in

435

macrophages, and had a higher bacterial load in mouse tissues, showing exacerbated

436

organ pathology. Macrophages infected with PPE11-expressing M. smegmatis

437

produced significantly greater amount of IL-1β, IL-6, TNF-α and an early decrease of

438

IL-10 along with higher levels of host cell death. These results indicate that PPE11

439

confers M. smegmatis with potential pathogenic properties.

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ACCEPTED MANUSCRIPT non-pathogenic fast-growing M. smegmatis mc2155.We found that expression of

440

Subcellular localization of PPE family proteins is often closely related to their

442

function. Previous studies have shown that PPE25, PPE57 and PPE68 are associated

443

with cell walls and may play a key role in pathogen-host interactions [33-35]. In this

444

study, we indicate that a member of the Mtb PE/PPE family proteins, PPE11

445

(Rv0453), is associated with the cell wall. The results of the growth curve show that

446

PPE11 has no significant effect on the growth of M. smegmatis. However, we found

447

that the overexpression of PPE11 improved the early survival of M. smegmatis in

448

infected macrophages in vitro and persistent for longer time in mouse organs (Liver,

449

spleen, lung). Our results are consistent with previous data that PE / PPE proteins

450

were involved in [32] and contribute to the survival and persistence of Mtb in

451

post-infection macrophages [31, 36]. In addition, the overexpression of PPE11 in M.

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smegmatis enhances the survival rate of M. smegmatis in the presence of lysozyme,

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hydrogen peroxide, and low pH (3 and 5), which further supports their role in

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ACCEPTED MANUSCRIPT modulating host innate immune response and imparting resistance to M. smegmatis

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against hostile environment.

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Cell death helps maintain intracellular homeostasis and provides protective immunity

458

against invasive pathogens [37]. Using the LDH assay, we found that THP-1

459

macrophages infected with M. smegmatis expressing PPE11 can cause more host cell

460

death than the control. This is similar to other possible virulence-related proteins in

461

Mtb. PE25 / PPE41 protein complexes, PE11 (Rv1169c) and PE_PGRS33 of the PE /

462

PPE family, induce necrotic cell death rather than apoptosis in macrophages [16, 38,

463

39]. PE_PGRS17 can cause host cell death and significantly higher TNF-α secretion

464

[40]. It is speculated that PPE11-induced host cell death is closely related to

465

tuberculosis virulence during infection, which may play a key role in the

466

pathogenesis of TB.

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Macrophages are essential for the infection and immunity of TB. In this study, we

469

found that M. smegmatis expressing PPE11 infected macrophages induced

470

significantly higher levels of pro-inflammatory cytokines (TNF-α, IL-6 And IL-1β),

471

while levels of anti-inflammatory cytokines (IL-10) decreased. This inflammatory

472

cytokine expression pattern shift may be related to the persistence and virulence of

473

Ms_PPE11 in the mouse infection model. TNF-α is necessary to control the growth of

474

mycobacteria and to form a protective granuloma reaction. The relative amount of

475

TNF-α in the infected site determines whether the cytokine is protective or

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Previous studies have found that PE-PGRS33 interacts with TLR-2 and induces TNF-

478

α secretion in macrophages to mediate the entry of Mtb into macrophages and

479

enhance virulence [28, 44]. Interleukin-1 (IL-1) is an important mediator of innate

480

immunity, and studies have revealed the interdependence between IL-1 and TNF- α

481

[45]. The pro-inflammatory pathway controlled by IL-1β has a contradictory effect,

482

preventing bacterial replication to prevent disease and promoting disease by causing

483

tissue damage [46, 47]. Mtb Rv3671c protein causes THP-1 cell necrosis, which may

484

be related to high levels of TNF-α and IL-1β expression [48]. Therefore, we

485

hypothesized that Ms_PPE11-induced host macrophage necrosis was caused by the

486

increased production of pro-inflammatory cytokines. ESAT-6 is an important

487

virulence factor for Mtb. We have found that ESAT-6 stimulates the production of

488

IL-6 in macrophages by STAT3 activation [49]. Mycobacterium can resist the innate

489

immune response through the upregulation of IL-6 production [50]. While

490

interactions between NK cell and CD11c + cell increase IL-6 production and drive

491

pathologic immune responses and the mortality associated with Mtb infection in

492

diabetic mice [51]. Similar findings have been found in other proteins in the PE / PPE

493

family, in which the destruction of Rv0485 (A transcription factor that regulates the

494

expression of PE13 and PPE18 in Mtb) leads to a decrease in TNF- α and IL-6

495

secretion levels in infected macrophages [52]. IL-10 is a key anti-inflammatory

496

mediator released during the host immune response against Mtb and plays an

497

important role in the proper balance between inflammation and immune pathology

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ACCEPTED MANUSCRIPT destructive. Overproduction of TNF-α can cause immune pathologic changes [41-43].

24

499

aseptic damage and an increase in the number of lesions [54]. The results suggest that

500

when TNF-α and IL-10 remain balanced, there may be a favorable granulomatous

501

environment in which the host cells, but shift of the balance can lead to different

502

infection outcomes [55]. Consistent with this observation, we found that there was a

503

wider range of tissue damage in the mouse tissues infected with Ms_PPE11 than

504

Ms_Vec, which may be associated with a higher level of TNF- α and IL-1β

505

production induced by PPE11 in macrophages. We observed significant inflammatory

506

cell infiltration and congestive oedema in the lung tissue of mice, and the aggregation

507

of macrophages. The appearance of epithelial cells and necrotic tissue is the key to

508

the formation of tuberculous granuloma. Our data further supports the notion that

509

excessive production of pro-inflammatory cytokines, decreased the production of

510

anti-inflammatory cytokines, and tissue damage may lead to the formation of

511

granulomas and the spread of bacterial infections.

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ACCEPTED MANUSCRIPT [53]. When the level of IL-10 is reduced, it will lead to an increase in the amount of

In summary, our data suggest that PPE11 expression changed the phenotype of

514

non-pathogenic M. smegmatis. Cell wall-related PPE11 affects the viability of

515

PPE11-expressing M. smegmatis-infected macrophages and its endurance in infected

516

mouse tissues. Furthermore, PPE11 expression causes a dynamic imbalance of

517

pro-inflammatory and anti-inflammatory cytokines and results in the corresponding

518

immunopathology of the tissue. These data strongly indicate that PPE11 may be

519

involved in complex pathogen-host interactions. However, further studies, such as

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ACCEPTED MANUSCRIPT homologous exchange knockout / silencing of the Mtb gene are required for the

521

determination of the mechanism by which PPE11 regulates the imbalance of

522

inflammatory factors, leading to the outcome of mycobacterial infections.

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ACCEPTED MANUSCRIPT 524

Conflict of interest

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The authors declare that they have no conflict of interest.

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Acknowledgements

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This work was supported by Grants from the Chinese National Key Project of

529

Infectious Disease (2012ZX10003008004).

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Ethical statement

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All the animal experiments were approved by and carried out under the guidelines of

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the Institutional Animal Ethics Committee of Sichuan University. Animals were

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sacrificed by cervical dislocation, and all efforts were made to minimize suffering.

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The following is the supplementary data related to this article:

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Supplementary data

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Figure legends

666

Fig. 1. The expression of PPE11 has no effect on the growth of recombinant M.

668

smegmatis. (A) PCR amplification of approximately 1557 bp of the Rv0453 gene +

669

6His in the Ms_PPE11 strain was identified by specific primers. (B) Ms_PPE11 and

670

Ms_Vec were grown at 37°C in M7H9 liquid medium to an OD600 of 0.6–1.0. Total

671

bacterial RNA was isolated and subjected to reverse transcription (RT)-PCR to detect

672

the expression of the ppe11 and aph genes. aph: a kanamycin resistance gene carried

673

by vector pMV261. (C) Mtb PPE11 was expressed in M. smegmatis and detected

674

using Western blotting. Cell lysates of Ms_Vec and Ms_PPE11 were subjected to

675

Western blot to determine the expression of the His-tagged PPE11 protein in M.

676

smegmatis by anti-His antibody. (D) Ms_Vec and Ms_PPE11 were grown in M7H9

677

medium supplemented with 0.05% Tween 80, 0.5% glycerol, with kanamycin (25

678

µg/ml). The OD600 was determined at an interval of 3 h. The results are

679

representative of three independent biological replicates.

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Fig. 2. PPE11 is associated with the Mycobacterium cell wall. Ms_PPE11 strains

682

expressing PPE11 were subjected to fractionation. PPE11 protein in different

683

fractions was detected by Western blotting using an anti-His antibody. The expression

684

of cytoplasm GroEL2 in each fraction was also detected by anti- GroEL2 antibody as

685

a control.

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686

31

688

survival of recombinant M. smegmatis within THP-1 macrophages. M. smegmatis

689

strains were treated with (A) lysozyme (2500 µg ml-1), (B) hydrogen peroxide, (C)

690

acidic pH (3 or 5) and (D) nitrosative stress (5 or 10 mM) for various times. One

691

hundred µl samples were removed and serially diluted, and the aliquots were plated

692

onto M7H10 agar plates. (E) PMA-differentiated THP-1 macrophages were infected

693

with Ms_Vec or Ms_PPE11 at an MOI of 10. At 4, 24, 48 and 72 h after infection, the

694

macrophages were washed and lysed using 0.5% Triton-X-100. Lysates were plated

695

on 7H10 medium containing 25 µg/ml kanamycin to determine the bacterial number.

696

Error bars represent (±SEM) three biological replicates. *p < 0.05, **p < 0.01. SEM,

697

standard error of the mean.

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ACCEPTED MANUSCRIPT Fig. 3. Sensitivity of M. smegmatis strains to stress conditions and Intracellular

Fig. 4. Cell death of THP-1 macrophages infected with recombinant M.

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smegmatis. Culture supernatants were collected from THP-1 cells infected at an MOI

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of 10:1 with Ms_PPE11 or Ms_Vec. The release of LDH as a measure of macrophage

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cell death was estimated by testing its activity in the culture supernatants. Error bars

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represent (±SEM) three biological replicates. *p < 0.05, **p < 0.01. SEM, standard

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error of the mean.

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Fig. 5. Determination of the production of TNF-α, IL-6, IL-1β and IL-10 in

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THP-1 macrophages infected by recombinant M. smegmatis. PMA-differentiated

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THP-1 macrophages were infected at an MOI of 10: 1 with Ms_Vec and Ms_PPE11, 32

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secretion of TNF-α (A), IL-6 (B), IL-1β (C) and IL-10 (D) was detected by ELISA.

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After 6 and 24 h of infection, the infected macrophages were collected and the

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transcription of TNF-α (E), IL-6 (F), IL-1β (G), and IL-10 (H) mRNA was detected

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by real-time PCR. The data presented here is the mean ± standard deviation (SD) of

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three independent experiments. * p <0.05, ** p <0.01 by student's two-tailed t test.

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ACCEPTED MANUSCRIPT respectively. Culture supernatants were collected at indicated intervals, and the

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Fig. 6. Persistence of M. smegmatis strains in the lung, liver and spleen of

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infected Balb/c mice. (A) Balb/c mice (n= 6) were infected intravenously with

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approximately equal numbers (5× 107) of Ms_Vec or Ms_PPE11 and sacrificed after

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3, 6 and 9 days post-infection for determination of the CFU in the (B) lung (C) liver

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and (D) spleen. Data represent the mean ± SD of 6 mice per group at each time point.

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*, p <0.05; **, p <0.01.

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Fig. 7. Organ pathology from infected Balb/c mouse. The lung (A), liver (B) and

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spleen (C) sections of Balb/c mice infected with 5 x 107 of Ms_Vec or Ms_PPE11

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were stained with haematoxylin and eosin (H&E) at day 7 post infection. Lung and

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liver slices in the box structure are shown at 40X magnification; liver slices with

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arrows to mark the spotty necrosis structure. Photographs of the representative

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sections from 2 mice are shown. Scale bar, 200 µm for lung, liver and spleen.

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Mice serum infected with Ms_Vec, Ms_PPE11 or PBS were collected at different

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time points (3, 6, 9 and 12 days) after infection. The secretion of TNF-α (A), IL-6 (B),

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IL-1β (C) and IL-4 (D) was detected by ELISA analysis. The data presented here are

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the mean ± standard deviation (SD) of three independent experiments. * p <0.05, ** p

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<0.01 by student's two-tailed t test.

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ACCEPTED MANUSCRIPT Fig. 8. Detection of serum cytokines in mice infected with recombinant strains.

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Fig. S1. Gross pathology of mouse spleens at day 7. BALB/c mice were infected

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with PBS, Ms_Vec and Ms_PPE11 strains via the lateral tail vein. The spleens were

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harvested at day 7 after infection.

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ACCEPTED MANUSCRIPT Table 1 Primers used in this study

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ppe11-F(i) ppe11-R(i) aph-F aph-R TNF-α-F TNF-α-R IL-6-F IL-6-R IL-10-F IL-10-R IL-1β-F IL-1β-R β-action-F β-action-R

Sequence(5′→3′) CGGGATCCATGACGAGCGCACTGATATGGATG CCAAGCTTTCAatggtgatggtgatgatgGTCCCCGCGGTCCCAA GTTC TGCCTGCCGTATCTGTCTTGGTTGACG CGAGTGTTGGGATTGGGCATTGGATT TCTTCCGACCATCAAGCA CACCGAGGCAGTTCCATA GGCGGTGCTTGTTCCT GCTACAGGCTTGTCACTCG GCCTTCGGTCCAGTTGCCTTCT TGCCAGTGCCTCTTTGCTGCTTT ACCTGGGTTGCCAAGCCTTGT GCTCCACGGCCTTGCTCTTGTTT TTCAGGCAGGCCGCGTCAGTTGT TGTGAGTCCCGGAGCGTGCAGTT GTGACGTTGACATCCGTAAAGA TGTGAGTCCCGGAGCGTGCAGTT

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Primers ppe11-F ppe11-R

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Highlights • To investigate the interaction and potential mechanism of PPE11 between pathogens and hosts, we heterologously expressed PPE11 in non-pathogenic, rapidly growing M. smegmatis strains. • Overexpression of the cell wall-associated protein, PPE11, can improve the viability of bacteria in the presence of multiple stressors. • Expression of PPE11 enhanced the early survival of M. smegmatis in macrophages and sustained a higher bacterial load in mouse tissues that showed exacerbated organ pathology.

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Macrophages infected with recombinant M. smegmatis produced significantly greater amounts of IL-1β, IL-6, TNF-α and an early decrease in IL-10 along with higher levels of host cell death. Similar cytokines changes were observed in the sera of infected mice. • The results indicate that PPE11 protein causes histopathological changes by disrupting the dynamic balance of the inflammatory factors and promoting host-cell death, indicating a potential role in the virulence of Mtb.