Role of Escherichia coli endopeptidases and dd -carboxypeptidases in infection and regulation of innate immune response

Accepted Manuscript Role of Escherichia coli endopeptidases and DD-carboxypeptidases in infection and regulation of innate immune response Sathi Mallick, Joyjyoti Das, Jyoti Verma, Samatha Mathew, Tapas K. Maiti, Anindya S. Ghosh PII:

S1286-4579(19)30055-3

DOI:

https://doi.org/10.1016/j.micinf.2019.04.007

Reference:

MICINF 4640

To appear in:

Microbes and Infection

Received Date: 3 October 2018 Revised Date:

21 April 2019

Accepted Date: 23 April 2019

Please cite this article as: S. Mallick, J. Das, J. Verma, S. Mathew, T.K. Maiti, A.S. Ghosh, Role of Escherichia coli endopeptidases and DD-carboxypeptidases in infection and regulation of innate immune response, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.04.007. 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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Title

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Role of Escherichia coli endopeptidases and DD-carboxypeptidases in infection and

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regulation of innate immune response

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Authors

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Sathi Mallicka, Joyjyoti Dasa, JyotiVermab, Samatha Mathewa, Tapas K. Maitia, Anindya S.

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Ghosha*

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Author affiliations

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a

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India, PIN-721302;

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b

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West Bengal, India, PIN-721302.

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*Correspondence: Anindya S. Ghosh, Professor, Department of Biotechnology, Indian

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Institute of Technology Kharagpur, West Bengal, India, PIN-721302.

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Phone: +913222283798

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Fax: +913222278707

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Email:[email protected]

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Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal,

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Advanced Technology Development Centre, Indian Institute of Technology Kharagpur,

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Abstract The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan

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recycling can also produce peptidoglycan fragments capable of activating an innate immune

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response in host. To investigate how these proteins in Enterobacteriaceae play a role to

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elicit/evade innate immune responses during infections, we deleted certain endopeptidases

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and DD-carboxypeptidases from E. coli CS109 and studied the viability of these mutants in

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macrophages. The ability of infected macrophages to exert oxidative killing, express surface

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activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses

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were elevated in macrophages infected with DD-carboxypeptidase mutants but reduced for

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endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated

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in macrophages infected with both mutant types. Overall, we have shown, under normal

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conditions endopeptidases have a tendency to elicit the immune response but their effect is

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suppressed by the presence of DD-carboxypeptidases. Conversely, DD-carboxypeptidases,

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normally, tend to reduce immune responses, as their deletions enhanced the same in

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macrophages. Therefore, we conclude that the roles of endopeptidases and DD-

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carboxypeptidases are possibly counter-active in wild-type cells where either class of

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enzymes suppresses each other's immunogenic properties rendering overall maintenance of

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low immunogenicity that helps E. coli in evading the host immune responses.

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Keywords:

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Penicillin-binding proteins; endopeptidase; DD-carboxypeptidase; Escherichia coli;

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infection; macrophages

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1.

Introduction

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Peptidoglycan (PG) is a strong mesh-like structure of the bacterial cell wall that acts as a

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stress-bearing element and provides the bacteria with its cell shape [1]. PG synthesis is

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catalyzed by a group of enzymes collectively termed as penicillin-binding proteins (PBPs)

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that

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endopeptidase (EPase) activities. PBPs are broadly classified into high and low-molecular-

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mass (LMM). In Escherichia coli, the LMM PBPs include PBP4, PBP5, PBP6, DacD, PBP7,

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AmpC, and AmpH. PBP4 and PBP7 are EPases while PBP5, PBP6, and DacD are DD-

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CPases. PBP4 was once believed to have both EPase and DD-CPase activities, though its

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EPase function is predominant [2–5]. Some LMM PBPs have partially overlapping functions

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that are dispensable in vitro [3,6]. However, loss of multiple LMM PBPs in combination with

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PBP5 can cause significant alterations in cellular morphology [3,6,7].

transpeptidase,

DD-carboxypeptidase

(DD-CPase)

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In addition to contribution in bacterial cell shape, PG also behaves as a potent

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immunogen that can elicit innate immune responses (IR) during bacterial infections [3]. Host

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phagocytic cells including macrophages play primary roles in innate immune defense against

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these bacterial pathogens [8]. A key factor in macrophage killing of ingested pathogens is the

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oxidative burst, involving synchronized production of reactive oxygen species (ROS) like

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superoxide ion, hydrogen peroxide and hydroxyl radicals, due to NADPH oxidase catalyzing

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the reduction of molecular oxygen [9]; and reactive nitrogen species (RNS) like nitric oxide

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(NO), catalyzed by inducible nitric oxide synthase (iNOS). NO subsequently reacts with

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superoxide forming peroxynitrite that is even more bactericidal [8,10–12]. Macrophages also

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undergo phagocytosis-induced apoptosis which can initiate an adaptive IR to pathogens like

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E. coli, Staphylococci or Streptococci [13]. PG recognition followed by macrophage

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activation takes place with the help of toll-like receptors- TLR2 expressed on the macrophage

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surface. TLR2-PG binding elicits intracellular signaling cascades that lead to production of

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cytokines (Tumor necrosis factor- TNFα, Interleukins- IL-1β, IL-6, IL-12, IL-18, IL-23), NO,

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leukotrienes and platelet-activating factors [8,14,15]. Prior studies have identified roles of high molecular mass (HMM) PBPs from Gram-

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positive bacteria in resistance to various innate immune defenses. PBP1a encoded by ponA

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gene in group B Streptococcus is an important factor, providing resistance against killing by

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the neutrophil antimicrobial peptides (α-defensins); whereas the ponA mutants were easily

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cleared by these phagocytes [16,17]. E. coli PBP1c, a membrane-anchored glycotransferase

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[18], in combination with a bacterial α2-macroglobulin YfhM, forms YfhM/PBP1c complex

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that functions in bacterial colonization, PG repair and periplasmic defense against host

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proteases, helping them in escaping the IRs [19]. PBP1c also mediates host-bacterial

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interactions in few other Gram-negative bacteria like Pasteurella multocida, Brucella

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abortus, and Salmonella enterica infections [19,20]. Few other studies in bacteria have linked

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the cell shape changes to evasion of host IRs; a transformation of bacillary E. coli to

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filamentous form helps it in evading macrophage phagocytosis [21]. A change in

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Helicobacter pylori helical to coccoid form helps in escaping the inflammatory responses of

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gastric epithelial cells [22,23]. Since LMM PBPs modify PG structure and cell shape, it has

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been speculated that they could possibly play a role in bacterial immune evasion [24].

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In this study, we attempt to find the LMM PBPs that could be responsible for modulating

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the PG, thereby eliciting the host innate IR by interaction with macrophage surface receptors,

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during E. coli infections. With this in mind, a series of single and multiple PBP deletion-

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mutants are constructed in an attempt to reveal this aspect of relevant lacunae in E. coli

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infections.

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2.

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2.1. Bacterial strains, plasmids, animal cell lines, culture media and chemicals

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Materials and methods

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The bacterial strains, plasmids, and their respective sources are mentioned in Table 1.

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Different media in bacterial growth and maintenance were Luria-Bertani broth & agar, and

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M9 agar (Hi-Media, India). Kanamycin (50 µg/mL) and ampicillin (50 µg/mL) were used for

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screening E. coli mutants whereas hygromycin (100 µg/mL) for maintaining pCHERRY8

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transformed E. coli. RAW264.7 murine macrophage cell lines (National Centre for Cell

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Sciences, India) were grown and maintained in Dulbecco’s Modified Eagle Medium

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(DMEM) with 10 % Fetal Bovine Serum (Gibco, Invitrogen life technologies, USA) and

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penicillin-streptomycin solution (100 µg/mL) at 37 °C in 5 % carbon dioxide. Anti-TLR2

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antibody, AlexaFluor 568 tagged-(donkey)-anti-mouse antibody and phycoerythrin-tagged

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major histocompatibility complex (MHC)-class II anti-mouse antibody were purchased from

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InvivoGen,

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immunosorbent assay (ELISA) cytoset kit and mouse TNF-α antibody pair were purchased

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from Invitrogen life technologies. MuLV reverse transcriptase kit for semi-quantitative real-

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time polymerase chain reaction (semi-qRT-PCR) was purchased from BioBharti Life Science

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Pvt. Ltd, India. Unless otherwise designated, all antibiotics, reagents, and chemicals were

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purchased from Sigma-Aldrich, and DNA-modifying enzymes from New England Biolabs,

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USA.

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2.2. Construction and curing of LMM PBP mutants

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E. coli CS109 single mutants- EPase mutants (∆PBP4 and ∆PBP7), DD-CPase

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mutants (∆PBP5, ∆PBP6, and ∆DacD) and multiple mutants- SM274, SK256, SM345,

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SM456, and SM56D were obtained from various sources mentioned in Table 1. The triple

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DD-CPase mutant SM36D was constructed by deleting dacD gene from the double DD-

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CPase mutant SK256. Here, transfer of kanamycin cassette ∆dacD::res-npt-res (KanR) from a

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CS109 ∆DacD strain was achieved by P1 transduction, as modified from Denome et al., 1999

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[6,25]. The constructed mutant was confirmed by its ability to grow in the presence of

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kanamycin (50 µg/mL) and by the absence of its PCR amplification using specific primers;

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forward primer (FP) 5’GCTTGAAACGCCGTCTTATTG3’ and reverse primer (RP)

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5’GCTCAGGCCTTATGGTGGAAATAA-3’.

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2.3. Microscopic examination of bacterial cell shape

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Overnight cultures of E. coli CS109, it’s single, and multiple PBP mutants were

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diluted to 1 % and grown to absorbance A600 ~ 0.4. Cell samples (5 µL) were placed on

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polylysine-coated slides and viewed at 100X magnification under a fluorescence microscope

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(Olympus IX71, Japan). The images were captured and analyzed using CellSens Dimension

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software [26,27].

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2.4. Bacterial Infection of Macrophages

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2.4.1. Infection conditions

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Overnight bacterial cultures were harvested at 8000 g for 5 minutes, washed twice and

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resuspended in phosphate buffer saline (PBS) to A600 ~ 0.2. RAW264.7 macrophages (2.5 x

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105 cells) in 6-well plates were treated with E. coli (CS109 and its LMM PBP mutants) at

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A600 ~ 0.2 to obtain a multiplicity of infection 200:1 [28] and allowed to stand undisturbed for

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60 minutes at 37 °C in 5 % carbon dioxide. This infection ratio was chosen to enable optimal

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infection within the experimental period for an ease of scrutiny through subsequent imaging

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experiments. Spent media was replaced to remove excess bacteria and likewise incubated

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further [10].

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2.4.2. Study of infection- Scanning electron microscopy (SEM)

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Samples of RAW264.7 cells treated with bacteria were prepared on cover glass,

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washed twice with PBS and fixed with 2.5 % glutaraldehyde solution for 15 minutes at room

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temperature. The cells were then dried using increasing concentrations of ethanol (50-100 %,

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5 minutes each), and desiccated for 30 minutes. Images of infected macrophages were

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captured at 1000X magnification through SEM (JSM5800, JEOL, Japan) [29].

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2.5. Determination of oxidative burst- ROS and RNS assays For determining the time of oxidative burst, RAW264.7 macrophages infected with

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CS109 were incubated for 24 hours and the levels of ROS and RNS produced during this

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event were estimated [10,30]. Upon finding the time of oxidative burst, the macrophages

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were subsequently infected with CS109 and its single LMM PBP mutants. ROS was detected

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qualitatively by fluorescence microscopy and quantitatively by flow cytometry (FACS

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Calibur flow cytometer, BD Biosciences) at 10,000 cell-counts. In both the instances, ROS

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generation was perceived using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA, 20 µM),

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a fluorescent dye that determines cellular ROS by green fluorescence (504/525 nm) [30].

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Additionally, a nuclear stain Hoechst 33342 (5 µg/mL) was used for capturing the

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fluorescence images.

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RNS was determined by distinguishing NO concentrations in the infected

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macrophage-supernatants, using Griess reagent A for 10 minutes in dark, followed by the

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addition of reagent B [10,30]. Readings were recorded at A540. Measures of NO produced

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were estimated by comparing the values with a standard sodium nitrite curve.

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2.6. Determination of bacterial viability within infected macrophages

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The bacterial viability was determined qualitatively by confocal microscopy and

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quantitatively by phagocytosis assay. For microscopy, CS109 and its single PBP mutants

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were transformed with pCHERRY8 for red fluorescence (587/610 nm) [31]. The infected

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macrophages with these transformed E. coli were stained with DCFDA for green

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fluorescence.

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To determine the counts of phagocytosed bacteria during oxidative burst, the RAW

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264.7 cells were subjected to lysis with 0.1 % Triton-X 100 and plated to determine the

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viable bacteria as colony forming units per mL of cell lysates [10,32,33]. The counts of

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bacteria before macrophage lysis (after washing the infected cells with PBS, pH 7.4) were

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also determined and negated from their counts after lysis, to avoid any errors from the un-

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internalized bacteria.

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2.7. Determination of macrophage activation: Surface expression of TLR2, MHC class II

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molecules, and TNF-α release

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As PG are potent TLR2 activators [34], these receptors on interaction with PBP

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mutants were determined by immunofluorescence microscopy following a protocol modified

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from Powers et al., 2006 [35]. After infection, the macrophages were fixed using 4 %

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paraformaldehyde and permeabilized with 0.1 % Triton-X-100, followed by 1 % bovine

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serum albumin treatment for an hour. Anti-TLR2 primary antibody binding was done for 6-8

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hours with subsequent AlexaFluor 568-tagged-donkey-anti-mouse secondary antibody

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binding for 30 minutes. The cells were washed 2-3 times with PBS and observed for TLR2 at

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603 nm under fluorescence microscope. MHC class II expression on macrophages were

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determined using phycoerythrin-tagged MHC class II anti-mouse antibody. These cells were

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analyzed by FACS at 10,000 cells counts and determined using CellQuest Pro software [30].

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TNF-α concentration in macrophage supernatants was measured using sandwich ELISA kit,

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following manufacturer’s guidelines.

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2.8. Determination of NFκB, iNOS and TNFα transcripts- Semi-qRT-PCR

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Infected macrophages were harvested and lysed using trizol. Total mRNA was isolated from

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the aqueous phase using isopropanol precipitation and employed for cDNA synthesis using

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MuLV reverse transcriptase kit. Specific primers were used to measure the levels of iNOS,

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TNF-α, nuclear factor-NFκB and β-actin (housekeeping gene) transcripts in the total cDNA

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by semi-qRT-PCR. Primers used were; β-actin- FP 5’GTTGGTTGGAGCAAACATCCC3’,

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RP 5’GAAGCAATGCTGTCACCTTCC3’; iNOS- FP 5’ACTACTACCAGATCGAGCCC3’

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and

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5’GTCCCCAAAGGGATGAGAAGT3’,

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NFκB-

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5’CAGTGCTGTCAGGGAGGAAG3’. The cDNA band-profile obtained thereby were

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analysed

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(http://imagej.nih.gov/ij/docs/index.html). Levels of respective targeted transcripts in

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infected macrophages were normalized against β-actin.

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2.9. Statistical analysis

5’ATTTCTTCAGAGTCTGCCCATTGCT3’;

5’GGCTACAGGCTTGTCACTCG3’

5’GAAGGAGATCATCCGCCAGG3’,

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All these experiments were executed at least thrice; the results were analyzed statistically

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by one-way ANOVA and considered significant at 95 % confidence levels with P value <

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0.05.

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3.

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3.1. Multiple LMM PBP mutants displayed aberrant cell shapes

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Consistent with the previous reports, CS109, its single and double LMM PBP mutants

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had similar morphologies, whereas the mutants lacking multiple LMM PBPs (including

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PBP5 deletion) had alterations in their cell shapes (Supplementary Fig. S1)[7].

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3.2. Macrophage infection with LMM PBP mutants

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To ensure if LMM PBP mutants could infect the macrophages, RAW264.7 cells were

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treated with CS109, its single EPase and DD-CPase mutants, and the infection was analyzed

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using SEM, in comparison with uninfected macrophages. The bacteria-treated macrophages

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displayed the presence of bacteria adhered to the macrophage surface. Many pseudopodial

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projections for bacterial phagocytosis were also detected on the infected cells. However,

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fewer pseudopodial projections appeared on uninfected control cells than their infected

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counterparts (Fig. 1a). These bacterial counts post-infections were subsequently determined

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by fluorescence microscopy and macrophage lysis.

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3.3. Oxidative Burst in macrophages occurred at 8th-hour post infection time

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On encounter of bacteria with macrophages, they internalize the bacteria by receptor-

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mediated phagocytosis. The phagocytes then produce an array of ROS and RNS, eventually

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killing these engulfed bacteria during the mechanism of oxidative burst [8,9,12]. Therefore,

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to understand this mechanism, RAW264.7 cells were infected with CS109 and generation of

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ROS and NO were determined over 24 hours, in time-lapse experimentations. Here, the peaks

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of both ROS and NO were observed at around 8th-hour post infection (hpi) with CS109 (Fig.

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1b). Similar results were observed for macrophages infected with LMM PBP mutants (data

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not shown). Therefore, unless otherwise specified, the remaining experiments were

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performed by considering 8th-hour as the peak for oxidative burst (8 hpi).

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3.4. Infection of macrophages with EPase mutants reduced while DD-CPase mutants

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increased the production of oxidative molecules

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As stated previously, ROS estimation in the infected macrophages (8 hpi) was

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performed using DCFDA. To check whether the PBP mutants exhibited any change in the

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pattern of oxidative killing, the RAW264.7 cells were infected with CS109, its EPase and

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DD-CPase mutants, and ROS production was detected by the emittance of green

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fluorescence. It was microscopically observed that macrophages infected with both the EPase

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mutants seemed to produce lower, while all the DD-CPase mutants produced higher

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fluorescence when compared to those infected with their parent (Fig. 2a).

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ROS generation in these macrophages was further confirmed semi-quantitatively by

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flow cytometry, where the mean fluorescence was directly proportional to the amount of ROS

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produced. The macrophages infected with both the EPase mutants- ∆PBP4 and ∆PBP7

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manifested significantly lower fluorescence (~ 70 and 60 % decrease, respectively) than the

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cells infected with CS109. However, the DD-CPase mutants differed from both the EPase

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mutants, wherein ∆PBP5, ∆PBP6, and ∆DacD mutants-infected macrophages recorded

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enhanced fluorescent signals (~ 20, 30 and 40 % increase respectively) when compared to the

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CS109-infected cells (Fig. 2b). These results for ROS generation by the infected

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macrophages were also supplemented with the generation of RNS.

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RNS detected in our study pertains to the NO produced by infected macrophages

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using Griess reagent. An increase in the color intensity of the reaction mixture containing

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spent media was estimated, which corresponded to an increase in NO produced. It was

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observed that on infection, the macrophages generated NO, following a pattern similar to

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ROS generation. The cells infected with both ∆PBP4 and ∆PBP7 mutants produced less NO

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than the cells infected with CS109 (~ 60 and 45 % reduction respectively). However, when

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infected with CS109, its ∆PBP5, ∆PBP6 and ∆DacD mutants, respectively, macrophages

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failed to show any significant difference in their NO levels (with ~ 5 % overall increase in

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each) (Fig. 2c).

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In addition to single mutants, the multiple LMM PBP mutants were also compared

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with CS109, to estimate the mutual ROS and NO levels produced by infected macrophages.

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Both ROS and NO generated by the double EPase mutant-SM274 infected macrophages

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remained significantly lower (~ 40 % decrease respectively) than the CS109-infected ones.

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ROS levels of the macrophages infected with double and triple DD-CPase mutants- SK256

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and SM36D were significantly higher (~ 20 and 30 % increase respectively) than CS109-

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infected cells. However, the NO concentrations on infection with these mutants remained

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similarly elevated as CS109 (~ 3-5 % insignificant increase). Also, macrophages infected

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with the triple, quadruple and quintuple mutants (SM345, SM456, and SM56D, respectively)

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showed significantly lesser ROS (~ 20, 15, 40 % decrease respectively) and NO (~ 30 %

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decrease in each) concentrations than when infected with CS109 (Fig.2d).

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3.5. Viability of EPase mutants increases while DD-CPase mutants decrease in macrophages

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during E. coli infections

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As a measure of bacterial pathogenesis, a significant part of the study dealt with

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determining the viability of phagocytosed bacteria within the macrophages during oxidative

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burst. To proceed with this approach, pCHERRY8 transformed-CS109 and single LMM PBP

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mutants were infected with RAW264.7 cells. Expression of the mCherry gene emitted red

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fluorescence at 610 nm; the protein, however, did not affect the bacterial virulence [31].

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Consequently, fluorescence microscopic images of the viable bacteria within macrophages

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were captured (Fig. 3a). The viable counts of the internalized bacteria were determined by

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lysing the infected macrophages.

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Macrophage lysis at 8 hpi, validates the capability of CS109 and its LMM PBP

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mutants to remain viable within RAW264.7 cells during oxidative burst. As observed, there

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was a continuous increase in the viable counts of ∆PBP4 and ∆PBP7 mutants (~ 82 and 140

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% increase respectively), while these counts for the DD-CPase mutants-∆PBP5, ∆PBP6 and

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∆DacD constantly reduced (~ 30, 60 and 80 % decrease respectively), compared to the counts

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of CS109 (Fig. 3b). The results were also confirmed for the viability of infecting LMM PBP

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mutants within macrophages at earlier stages of infection (2 hpi), by gentamicin assay

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(protocol modified from Baumgart et al., 2007) [36] that showed similar survival patterns,

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(Supplementary Fig. S2). The survival of LMM PBP mutants was thereafter studied with a

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ROS inhibitor N-acetyl-L-cysteine (NAC) at 8 hpi [37]. Herein, an increase in survival of the

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PBP mutants was observed within NAC-treated macrophages, confirming the decrease in

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bacterial viability, observed previously due to oxidative killing (Supplementary Fig. S3).

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3.6. Expression profiling of surface receptors and release of cytokines on macrophage

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infection with E. coli LMM PBP mutants

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The events of oxidative burst and bacterial viability during innate IR directly correlate

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to macrophage activation. The activation leads to an increase in transcription and translation

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of certain genes, with respect to nuclear factors and surface receptors. A consequent release

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of various cytokines results in bacterial clearance and subsequent interaction with cells of the

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adaptive immune system in E. coli infections.

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3.6.1. TLR2 surface expressions on macrophages decrease on infection with EPase mutants

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and increases with DD-CPase mutants

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On bacterial infections, the macrophages receive a strong activation stimulus from PG

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interaction. PG, a pathogen-associated molecule triggers the surface expression of TLR2, a

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pattern-recognition molecule on macrophage plasma membrane [14,34,38], the expression of

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which is at basal levels otherwise. Therefore, the TLR2 expression on macrophage surface

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were estimated qualitatively by immuno-fluorescence microscopy post infection with CS109,

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its single EPase, and DD-CPase mutants. If expressed, TLR2 on macrophages produced an

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orange fluorescence due to final binding with AlexaFluor 568 tagged-secondary anti-mouse

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antibody. Once again, the fluorescence produced by macrophages, infected with both the

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EPase mutants- ∆PBP4 and ∆PBP7, appeared to be significantly lesser (~ 63 and 65 %

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reduction) than the parent-infected cells. Conversely, infection with all three DD-CPase

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mutants, led to an elevated fluorescence, similar to the cells infected with their parent (no

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significant difference) (Fig. 4a and 4b). This may depict lower stimulation of macrophages

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when infected with the EPase mutants that bring about fewer TLR2 surface expressions than

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infection with DD-CPase mutants.

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3.6.2. EPase mutants failed to increase the levels of MHC class II molecules on macrophages

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unlike the DD-CPase mutants

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Like the surface expression of TLR2, expression of MHC class II molecules is also a

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measure of IR on stimulation with PG. When the RAW264.7 cells were infected with CS109

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and its LMM PBP mutants, the infection with EPase mutants- ∆PBP4 and ∆PBP7 had

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significantly lower expressions of MHC class II molecules (~ 40 and 15 % decrease), while

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that with DD-CPase mutants- ∆PBP5, ∆PBP6, and ∆DacD demonstrated a significant

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increase (~ 35, 30 and 20 % higher) in these molecules than the CS109-infected cells (Fig.

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4c).

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3.6.3. EPase mutants restrained the release of TNF-α from macrophages

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Production of TNF-α by the infected macrophages was estimated by ELISA. On

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comparison, TNF-α concentrations had significantly reduced after the macrophages were

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infected with EPase mutants- ∆PBP4 and ∆PBP7 (~ 40 and 50 % reduction) than when

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infected with CS109 or its single DD-CPase mutants. However, the levels of TNF-α remained

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similarly elevated in the macrophages infected with the three single DD-CPase mutants, and

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CS109 (an insignificant 1 % increase in all three) (Fig. 4d).

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3.8. Levels of NFκB, iNOS and TNF-α transcripts in CS109 and its LMM PBP mutants

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infected macrophages remained nearly similar

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Interaction of the TLR2 with PG leads to activation of the macrophages which

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successively increases the transcription and translation of its essential genes concerning

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oxidative killing. The levels of NFκB, iNOS and TNF-α transcripts in infected macrophages,

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were therefore determined by semi-qRT-PCR (Fig. 5). These transcripts in macrophages

332

infected with single DD-CPase mutants remained constantly higher or similar to the CS109-

333

infected ones. The cells infected with ∆PBP5 and ∆PBP6 mutants both had a significant

334

increase in NFκB transcripts (~ 20 % increase in each mutant) and iNOS transcripts (~ 20 and

335

40 % increase, respectively) than the CS109-infected cells.

336

macrophages infected with parent and mutant E. coli showed a nearly similar results.

337

However, the macrophages infected with ∆DacD mutant exhibited NFκB transcript levels

338

like in cells infected with CS109; and significantly higher iNOS and TNF-α transcripts (~ 30

339

% increase in each transcript) than the ones infected with CS109. Whereas, in contrast to the

340

in vitro experiment results, these transcripts in macrophages on infection with the EPase

341

mutants also remained at a higher end like in CS109-infected cells. Specifically, the ∆PBP4

342

mutant-infected macrophages had all the NFκB, iNOS, and TNFα transcripts significantly

343

elevated (~ 20, 63 and 25 % increase respectively). However, the macrophages infected with

344

the ∆PBP7 mutant had a significant increase in iNOS transcripts (~ 40 % increase) while the

345

NFκB and TNF-α transcripts remained similar to that of CS109-infected ones.

346

4.

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For TNFα transcripts,

The pattern of IR remains similar upon macrophage infection with E. coli LMM PBP

348

mutants of a particular group. The oxidative burst within these cells occurs at 8 hpi with

349

CS109. When macrophages were infected with the EPase mutants- ∆PBP4 and ∆PBP7, the

350

overall response in terms of ROS and RNS production, expression of TLR2 and MHC class II

351

molecules and secretion of TNFα for innate immune activation and signaling to adaptive

352

immune system cells, remains lower. Conversely on infection with the DD-CPase mutants-

353

∆PBP5, ∆PBP6, ∆DacD, these responses are nearly equal or higher than the responses in

354

macrophages infected with CS109. However, the internalized EPase mutants show an

355

increased survival within macrophages than the DD-CPase mutants which is inversely

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proportional to the kind of responses they produce during oxidative killing and macrophage

357

activation. Higher the response, higher is the ability of macrophages to kill the infecting

358

bacteria, and lower is the bacterial capability to survive within these cells [10,33]. On bacterial infections, the PG-activated macrophages follow the TLR2/IKβ

360

kinase/NFκB pathway for activation [14], which leads to increased transcription and

361

subsequent translation of various genes. Here, the NFκB, iNOS, and TNF-α transcripts in

362

macrophages remain similarly elevated on infection with both CS109 and its DD-CPase

363

mutants, like the production of oxidative molecules, surface receptors, and cytokines. When

364

macrophages detect pathogen-associated molecules during bacterial infections, its NFκB

365

expressions are triggered that up-regulates the transcriptional levels of subsequent genes, for

366

increased expression of their respective products, required during innate IRs. Nevertheless, an

367

increase in the transcripts is also seen in EPase mutants-infected cells, though the

368

concentrations of their expressed product molecules remain lowered; the reason for this is yet

369

unknown. Many bacteria regulate the production of toxic molecules from macrophages, by

370

expression of their desired genes. During infections, Leishmania donovani and L. Major

371

suppress iNOS expressions in macrophages for lowered NO production, by increasing the

372

concentrations of phosphotyrosine phosphatases and glycoperoxidases in these cells.

373

Similarly, E. coli and Salmonella typhimurium induce their SoxRS and OxyR regulons to

374

reduce the final concentrations of NO in macrophages [11]. The EPase mutants, therefore, are

375

similarly suspected to be involved in negative regulation, to block the intermediates of

376

specific pathways and impede toxic end product formation in macrophages, thereby

377

inhibiting an innate IR. In summary, E. coli in its natural form probably may suppress the

378

expression of its EPases (PBP4 and PBP7), to help itself survive within host during

379

infections.

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Events of the macrophage responses towards bacterial infections can be correlated with

381

various mechanisms of bacteria and their ability to either survive the immunological attack or

382

to perish thereby. PG shedding is one such mechanism among Gram-positives and few Gram-

383

negatives like E. coli, Neisseria gonorrheae, Bordetella pertussis, and Shigella flexneri, by

384

which the dividing bacteria release muramyl peptides into their environment through the

385

catalytic activity of their PG hydrolyzing enzymes [38]. Since PG acts as a strong

386

immunogen, the muramyl peptides too are expected to behave analogously. They interact

387

with TLR2 and activate the macrophages via TLR2/IKβ kinase/NFκB pathway [14,39], to

388

trigger a cascade of immunological reactions. However, there are certain other bacterial

389

agents including the different lipoproteins, outer membrane proteins, zymosans, etc. that also

390

act as TLR2 agonists [18,40]. Outer membrane lipoproteins, LpoA and LpoB in E. coli are

391

known to regulate the functioning of transglycosylase and transpeptidase activities of HMM

392

PBPs in PG synthesis, PBP1a and PBP1b, respectively [41]. The way, interactions with

393

HMM PBPs is a well-defined phenomenon for TLR2 activation, a rare participation of any

394

lipoprotein influencing the enzymatic activity of the LMM PBPs in PG synthesis is

395

recognized so far. The one recent example of NlpI regulating PBP4 is explained ahead [42].

396

However, an additional possibility of the lipoproteins to be involved in TLR2 responses with

397

LMM PBPs might not be denied. A further detailed study might help in finding such

398

unanswered questions in relation to correlating the effects of lipoproteins and PG in TLR2

399

activation via LMM PBPs. Therefore, so far as PG is concerned, it may be speculated that for

400

escaping from the IRs and surviving within the host, the bacteria may regulate their PG-

401

hydrolases to constrain PG shedding [24].

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402

Of the 13 different PG-hydrolases in E. coli [18], the EPases- PBP4 and PBP7 are

403

confirmed for their roles in the shedding mechanism [5]. They break the peptide bonds

404

between two consecutive PG-polymers and release short muramyl peptides that are recycled

18

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back into the growing PG of dividing bacteria. DD-CPases are also PG hydrolases but are not

406

known to be involved in any such process. Pertaining to the experimental evidence, in such a

407

situation, bacteria could choose to suppress the expression of their EPases, specifically PBP4,

408

to help them endure the IRs and outlive the challenge to thrive within their host. Until

409

recently, an outer membrane lipoprotein NlpI (mentioned previously), is found to negatively

410

regulate the enzymatic activity of E. coli PBP4, suppress its function and reduce the release

411

of muramyl peptides [42]. Hence, it can be said that the negative regulation of PBP4 by E.

412

coli increases its PG-crosslinks for a stronger cell wall, with increased stress bearing tenacity,

413

which helps the bacterium to evade the host IRs.

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Arresting PG shedding is just not sufficient for the bacteria to lower the host IRs.

415

Factors like the bacterial shape and its aspect ratio are also involved that inhibits bacterial

416

phagocytosis by macrophages [21]. The bacterial shape depends upon its cell wall, the pattern

417

of its synthesis and also the cell wall synthesizing enzymes like PBPs. H. pylori bring about

418

conversion between its spiral to coccoid form by employing AmiA protein [22,23]. This

419

morphological change helps it to escape the nucleotide-binding oligomerization domain-

420

containing protein1- Nod1/NFκB signaling pathway and a decrease in the subsequent release

421

of IL-8 from gastric epithelial cells for inflammatory responses [23]. PBP5 is an important

422

enzyme for maintaining the bacterial shape, deletion of which along with two other LMM

423

PBPs renders the cell aberrant [7,43]. MreB, another protein which enables the cell to

424

maintain its shape; though unlike PBP5 it is an essential protein, for maintaining the bacterial

425

cytoskeleton [27]. Bacterial shape change towards escaping the phagocytic activity of

426

macrophages has also been demonstrated in E. coli. A change from its bacillary to

427

filamentous form is attained by inhibition of cell septation, increasing its aspect ratio several

428

times than the bacillary state (aspect ratio 3); which in turn, facilitates the deformed E. coli to

429

evade its host responses [21]. An example of this can be demonstrated in uropathogenic E.

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coli wherein expression of its SulA protein leads to filamentation in response to the

431

phagocytic effect of neutrophils. There are numerous illustrations of other factors involved in

432

different bacteria that lead to their morphology change for avoiding internalization or survival

433

within phagocytes. These include filamentation in Mycobacterium tuberculosis, Proteus

434

mirabilis, Salmonella typhimurium, Shigella flexneri and biofilms formed by filamentous

435

forms of Haemophilus influenza and Legionella pneumophila. [44].

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Therefore, to find a correlation between the IRs, cell shape, and PBPs, single and

437

multiple E. coli LMM PBP mutants are studied together for ROS and NO production during

438

macrophage infections. The single and double EPase mutants- ∆PBP4, ∆PBP7, and SM274

439

illustrate a lower response than parent CS109 when infected with macrophages.

440

Alternatively, the macrophages infected with single and double DD-CPase mutants- ∆PBP5,

441

∆PBP6, ∆DacD, and SK256 show an increased level of these responses on infection. In both

442

instances, the single and double mutants display intact shapes similar to CS109, without

443

exhibiting any notable changes in terms of shape defects. However, shape defects are

444

remarkably observed otherwise in the multiple mutants with three or more LMM PBP

445

deletions (including PBP5 deletion)- SM345, SM36D, SM456 and SM56D [7,43]. On

446

infection of the deformed SM345, SM456 and SM56D mutants (with both EPases and DD-

447

CPases deletions), the macrophages are observed for a reduced oxidative killing. Thus, the

448

shape deformation and PBP deletions can be correlated to IRs, where aberrant shape prevents

449

the bacteria from getting cleared by macrophage phagocytosis [21]. However, although

450

distorted, the DD-CPase triple mutant- SM36D leads to elicitation of a higher response in

451

macrophages. Herein, the PBPs once again illustrate their importance, suggesting that it’s just

452

not the bacterial shape, but also the LMM PBPs that are crucial in evading the IRs. As long as

453

the multiple mutants (triple deletion onwards) are included with EPase deletions, response of

454

the infected macrophages remain lowered. In contrast, on infection with SM36D, the

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responses are high and similar to CS109. It proves that bacterial shape change is not the only

456

phenomenon affecting the innate IR, but LMM PBPs too that together play a synergistic role;

457

and it is the DD-CPases in E. coli that are speculated to suppress the immunological activity

458

of EPases to help the bacterium in evading the host IRs.

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Therefore, based on the results obtained, it can be concluded that normally during E.

460

coli infections, the EPases elicit innate IRs whereas the DD-CPases help in limiting them.

461

Hence, it is the combinatorial effort of LMM PBPs which facilitate E. coli in invading the

462

immune system. Where the EPases show a clearer picture, the role of DD-CPase in

463

controlling the IRs remains a mystery. A study of infection in other aspects of the innate

464

immune system may thus help in lighting some areas of their function.

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Acknowledgments

We thank Santoshi Nayak for the help in conducting the experiments. We thank Dr.

468

David E. Nelson, Indianapolis, Indiana, USA for his constructive suggestions. The work is

469

supported by a grant from the Department of Biotechnology (DBT), Govt. of India to

470

Anindya S. Ghosh [File No. BT/PR8539/BRB/10/1235/2013]. Sathi Mallick is supported by

471

a fellowship for doctoral studies from the Council of Scientific and Industrial Research [File

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NO: 09//081(1096) 2010-EMR-I].

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Conflicts of interest None to declare

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478 479

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activity. PNAS 2016;113:4788–4793. [42] Schwechheimer C, Rodriguez DL, Kuehn MJ. NlpI-mediated modulation of outer membrane

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Legends of figures

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Fig. 1: Macrophage infection and oxidative burst over 24 hours (a) Scanning electron

598

microscopic (SEM) analysis of macrophages infected with E. coli LMM PBP mutants.

599

(i)Uninfected macrophages treated with PBS; (ii-vii) RAW264.7 cells infected with E. coli

600

CS109 and its single mutants ∆PBP4, ∆PBP7, ∆PBP5, ∆PBP6, ∆DacD respectively; here the

601

black arrows point to the bacteria adhered to macrophages. (b) Oxidative burst in

602

RAW264.7 macrophages peaks at 8 hours post-infection with the E. coli CS109. This

603

process was traced over 24 hours time period and represented in terms of (i) ROS and (ii) NO

604

production.

605

Fig. 2: Oxidative killing in RAW264.7 macrophages infected with E. coli LMM PBP

606

mutants. (a) ROS production in RAW264.7 cells on infection with the EPase and DD-

607

CPase mutants; (i) phase contrast and (ii) fluorescent images of RAW264.7 cells stained

608

with DCFDA (green) and counterstained with HOECHST33342 (blue). Uninfected

609

macrophages treated with (I) PBS exhibiting least fluorescence (negative control), and (II) 10

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µg/mL LPS exhibiting increased fluorescence (positive control) (III) macrophages infected

611

with parent E. coli CS109; (IV-V) infected macrophages treated with the EPase mutants-

612

∆PBP4 and ∆PBP7 respectively emitting lesser fluorescence than the parent; (VI-VIII) cells

613

infected with the DD-CPase mutants- ∆PBP5, ∆PBP6 and ∆DacD respectively emitting

614

higher fluorescence than the parent. (b) ROS determination in the infected RAW264.7

615

cells by flow cytometry. (i-ii) macrophages infected with the EPase mutants- ∆PBP4 and

616

∆PBP7emitting lower fluorescence than the parent-infected cells, shown along with

617

comparative histograms (iii-iv) macrophages infected with DD-CPase mutants (∆PBP5,

618

∆PBP6, and ∆DacD)- infected cells emitting higher fluorescence compared to parent-infected

619

cells, shown along with their comparative histograms. (c) The release of NO by infected

620

macrophages. NO released by the (i) E. coli EPase and (ii) DD-CPase mutants-infected

621

macrophages respectively. The macrophages infected with both the EPase mutants (∆PBP4

622

and ∆PBP7) produced significantly lower concentrations of NO, whereas when infected with

623

the DD-CPase mutants (∆PBP5, ∆PBP6, and ∆DacD) the NO levels remained similar (with

624

no statistical difference) to the macrophages infected with the parent E. coli CS109 strain. (*)

625

implies statistically significant difference at P value < 0.05. (d) Combinatorial effects of the

626

EPase and DD-CPase deletions on oxidative killing in macrophages during E. coli

627

infections. RAW264.7 macrophages were studied for the ROS (i) and RNS (ii) when

628

infected with PBP single and multiple mutants. (i) Macrophages infected with both the EPase

629

single mutants ∆PBP4 and ∆PBP7 and the double mutant SM274 (∆PBP 7, 4) showed ROS

630

production values lesser than the parent CS109-infected macrophages. The DD-CPase single

631

mutants (∆PBP5, ∆PBP6, and ∆DacD mutants), double mutant SK256 (∆PBP5, 6) and triple

632

mutant SM36D (∆PBP5, 6, DacD) infected macrophages showed higher levels of ROS

633

production. However, macrophages infected with the sequential-multiple mutants also

634

showed lower ROS production in comparison with the CS109-infected macrophages. (ii) The

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results in terms of RNS production on infection with the EPase single and double mutants

636

and cumulative effect of the multiple mutants SM345 (∆PBP 7, 4, 5), SM456 (∆PBP 7, 4, 5,

637

6) and SM56D (∆PBP 7, 4, 5, 6, DacD) remained similar to (i); but, when infected with the

638

DD-CPase single, double and triple mutants, the macrophages produced RNS similar to

639

CS109-infected cells. (*) implies a statistically significant difference in the results, between

640

the parent and all the respective mutants, infected macrophages at P value < 0.05.

641

Fig. 3: Bacterial viability within the infected macrophages. (a) Confocal micrographs of

642

RAW264.7 cells infected with the pCHERRY8- transformed E. coli LMM PBP mutants.

643

(I) Control uninfected macrophages treated with PBS; (II) RAW264.7 macrophages infected

644

with parent E. coli CS109; (III-VII) macrophages infected with the EPase mutants- ∆PBP4

645

and ∆PBP7, and DD-CPase mutants- ∆PBP5, ∆PBP6, and ∆DacD respectively. For all the

646

images, panel (i) represents phase contrast images; (ii) shows the infected macrophages

647

stained with DCFDA (green); panel (iii) shows the pCHERRY8- transformed bacteria (red);

648

panel (iv) merged images of (ii & iii). (b) Macrophage lysis and count of viable LMM

649

PBP mutants at 8 hours post-infection time. On lysis of the infected macrophages, (i) both

650

the EPase mutants showed a significantly increased survival with more colony count as

651

compared to their parent E. coli CS109; whereas, (ii) all the three DD-CPase mutants showed

652

counts that were gradually decreasing from that of the E. coli CS109. (*) implies a

653

statistically significant difference between the parent and all respective mutants involved, at

654

P value < 0.05.

655

Fig. 4: Expression of surface receptors and release of cytokines. (a) Surface expression

656

of TLR2 on infected RAW264.7 macrophages. The orange fluorescence of the

657

macrophages demonstrates TLR2 expression when bound to AlexaFluor 568-tagged

658

antibodies. Panel (i) represents phase contrast and panel (ii) represents immunofluorescent

659

images of the same. (I) Cells treated with PBS produced the least fluorescence (negative

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control) whereas (II) Cells activated with 50 µg/mL zymosan (positive control) emitted a

661

higher intensity of fluorescence. (III) Macrophages infected with E. coli CS109; (IV-V)

662

macrophages treated with the EPase mutants- ∆PBP4 and ∆PBP7 had lower TLR2 surface

663

expression, therefore emitted lesser fluorescence, however, (VI-VIII) macrophages infected

664

with the DD-CPase mutants - ∆PBP5, ∆PBP6 and ∆DacD, and their parent CS109 emitted an

665

increased fluorescence due to increased levels of the TLR2 expressions on their surfaces. (b)

666

Graphical representation of TLR2 surface expression on macrophages based on their

667

fluorescence intensities. Fluorescence intensities emitted from (i) the macrophages infected

668

with EPase mutants had decreased, while (ii) the macrophages infected with DD-CPase

669

mutants were elevated like in the CS109-infected cells, corresponding to the respective levels

670

of TLR2 surface expressions. (c) Determination of MHC class II molecules on the infected

671

RAW264.7 cells by flow cytometry. (i-ii) The macrophages infected with EPase mutants-

672

∆PBP4 and ∆PBP7 emitting lower fluorescence than the parent CS109 due to significantly

673

lower levels of MHC class II expression, represented along with a comparative histogram;

674

(iii-iv) Macrophages infected with the DD-CPase mutants- ∆PBP5, ∆PBP6, and ∆DacD,

675

emitted significantly higher fluorescence than the parent CS109-infected cells, due to an

676

increase in the surface expression of their MHC class II molecules, which is shown along

677

with its comparative histogram. (d) The release of TNFα by infected macrophages. The

678

macrophages (i) infected with both the EPase mutants- ∆PBP4 and ∆PBP7 produced

679

significantly lower concentrations of TNFα, whereas (ii) when infected with the DD-CPase

680

mutants- ∆PBP5, ∆PBP6, and ∆DacD, these levels remained similar to the macrophages that

681

were infected with the parent E. coli CS109 strain. (*) implies statistically significant

682

difference at P value < 0.05.

683

Fig. 5: Analysis of iNOS, TNFα and NFκB transcripts from infected macrophages by

684

semi-qRT-PCR. Levels of iNOS, TNFα and NFκB transcripts isolated from the

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macrophages-infected with (a) EPase mutants and (b) DD-CPase mutants respectively,

686

normalized against β-actin, in (i) band images and (ii) graphical representation of transcript

687

levels. (a) In case of macrophages infected with EPase mutant ∆PBP4, the levels of these

688

transcripts remain significantly high; whereas for the ∆PBP7 mutant, it remains similar for

689

NFκB and TNFα transcripts but higher for transcripts of iNOS, in comparison with the cells

690

infected with the parent CS109. (b) On infection with the DD-CPase mutants; the mRNA

691

levels of macrophages infected with ∆PBP5 and ∆PBP6 mutants were significantly high for

692

NFκB and iNOS but similar for TNFα, as compared to cells infected with the parent.

693

However, on infection of the ∆DacD mutants, these macrophages had their NFκB transcript

694

levels similar, whereas the levels of iNOS and TNFα transcripts were significantly higher

695

than the CS109-infected macrophages. (*) implies a statistically significant difference

696

between transcripts of the parent and respective mutants infected macrophages at P value <

697

0.05.

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ACCEPTED MANUSCRIPT Table 1: List of bacterial strains and plasmids

Deleted PBP

Source

W1485 rpoS rph

None

[6]

CS109 ∆PBP4

W1485 rpoS rph ∆dacB

PBP 4

[6]

CS109 ∆PBP7

W1485 rpoS rph ∆pbpG

CS109 ∆PBP5

W1485 rpoS rph ∆dacA

CS109 ∆PBP6

W1485 rpoS rph ∆dacC

CS109 ∆DacD

W1485 rpoS rph ∆dacD

PBP 7

[6]

PBP5

[6]

PBP6

[6]

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CS109

RI PT

Genotype featured

Strain/Plasmid

Dac D

[25]

PBP4, PBP7

[27]

W1485 rpoS rph ∆pbpG ∆dacB

SM345

W1485 rpoS rph ∆pbpG ∆dacB ∆dacA

PBP4, PBP7, PBP5

[27]

SM456

W1485 rpoS rph ∆pbpG ∆dacB ∆dacA

PBP4, PBP7,

[27]

∆dacC

PBP5, PBP6

W1485 rpoS rph ∆pbpG ∆dacB ∆dacA

PBP4, PBP7,

TE D ∆dacC ∆dacD

PBP5, PBP6, DacD

SK256

W1485 rpoS rph ∆dacA ∆dacC

PBP5, PBP6

SM36D

W1485 rpoS rph ∆dacA ∆dacC ∆dacD

S17-1/λpir

EP

SM56D

M AN U

SM274

pCHERRY8

This work

PBP5, PBP6, DacD This work

recAthi pro hsdR [res2 mod1]

None

[25]

Suicide plasmid—containing a cloned

None

[6]

None

[31]

AC C

pJMSB8

[27]

parA resolvase gene

Plasmid with fluorescent gene mCherry cloned in pSMT3-rpsA vector

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