Role of Glycolipids in the Pathogenesis of Enterococcus faecalis Urinary Tract Infection

Background

After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells.

Results

In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.

Conclusions

Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.     

Bacteriocin synthesis in uropathogenic and commensal <Emphasis Type="Italic">Escherichia coli</Emphasis>: colicin E1 is a potential virulence factor

Background  

Bacteriocin production is an important characteristic of E. coli strains of human origin. To date, 26 colicin and 9 microcin types have been analyzed on a molecular level allowing molecular detection of the corresponding genes. The production incidence of 29 bacteriocin types and E. coli phylogroups were tested in a set of 361 E. coli strains isolated from human urinary tract infections (UTI) and in 411 control strains isolated from feces of patients without bacterial gut infection.     

<Emphasis Type="Italic">Coxiella burnetii</Emphasis> Nine Mile II proteins modulate gene expression of monocytic host cells during infection

Background  

Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection.     

Common and specific genomic sequences of avian and human extraintestinal pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> as determined by genomic subtractive hybridization

Background  

Suppression subtractive hybridization (SSH) strategy was used with extraintestinal pathogenic Escherichia coli (EXPEC) that cause avian colibacillosis (avian pathogenic E. coli or APEC) and human urinary tract infections (uropathogenic E. coli or UPEC) to determine if they possessed genes that were host and/or niche specific. Both APEC and UPEC isolates were used as tester and driver strains in 4 different SSHs in order to obtain APEC- and UPEC-specific subtraction fragments (SFs).     

Reduced evolvability of <Emphasis Type="Italic">Escherichia coli</Emphasis> MDS42, an IS-less cellular chassis for molecular and synthetic biology applications

Background  

Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential.     

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Background  

The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.     

Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

A Novel Role for D-Alanylation of Lipoteichoic Acid of Enterococcus faecalis in Urinary Tract Infection

Background

Enterococci are the third most common cause of healthcare-associated infections, which include urinary tract infections, bacteremia and endocarditis. Cell-surface structures such as lipoteichoic acid (LTA) have been poorly examined in E. faecalis, especially with respect to urinary tract infections (UTIs). The dlt operon is responsible for the D-alanylation of LTA and includes the gene dltA, which encodes the D-alanyl carrier protein ligase (Dcl). The involvement of LTA in UTI infection by E. faecalis has not been studied so far. Here, we examined the role of teichoic acid alanylation in the adhesion of enterococci to uroepithelial cells.

Results

In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔdltA mutant colonizes uroepithelial surfaces more efficiently than wild type bacteria. We also demonstrated that this mutant adhered four fold better to human bladder carcinoma cell line T24 compared to the wild type strain. Bacterial adherence could be significantly inhibited by purified lipoteichoic acid (LTA) and inhibition was specific.

Conclusion

In contrast to bacteraemia model and adherence to colon surfaces, E. faecalis 12030ΔdltA mutant colonized uroepithelial surfaces more efficiently than wild-type bacteria. In the case of the uroepithelial surface the adherence to specific host cells could be prevented by purified LTA. Our results therefore suggest a novel function of alanylation of LTA in E. faecalis.     

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

Background  

Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

The Natural Antimicrobial Carvacrol Inhibits Campylobacter jejuni Motility and Infection of Epithelial Cells

Background

Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni.

Methods/Principal Findings

Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function.

Conclusions/Significance

Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.     

Mucin Dynamics in Intestinal Bacterial Infection

Background

Bacterial gastroenteritis causes morbidity and mortality in humans worldwide. Murine Citrobacter rodentium infection is a model for gastroenteritis caused by the human pathogens enteropathogenic Escherichia coli and enterohaemorrhagic E. coli. Mucin glycoproteins are the main component of the first barrier that bacteria encounter in the intestinal tract.

Methodology/Principal Findings

Using Immunohistochemistry, we investigated intestinal expression of mucins (Alcian blue/PAS, Muc1, Muc2, Muc4, Muc5AC, Muc13 and Muc3/17) in healthy and C. rodentium infected mice. The majority of the C. rodentium infected mice developed systemic infection and colitis in the mid and distal colon by day 12. C. rodentium bound to the major secreted mucin, Muc2, in vitro, and high numbers of bacteria were found in secreted MUC2 in infected animals in vivo, indicating that mucins may limit bacterial access to the epithelial surface. In the small intestine, caecum and proximal colon, the mucin expression was similar in infected and non-infected animals. In the distal colonic epithelium, all secreted and cell surface mucins decreased with the exception of the Muc1 cell surface mucin which increased after infection (p<0.05). Similarly, during human infection Salmonella St Paul, Campylobacter jejuni and Clostridium difficile induced MUC1 in the colon.

Conclusion

Major changes in both the cell-surface and secreted mucins occur in response to intestinal infection.     

Diversity of bacterial manipulation of the host ubiquitin pathways

Ubiquitination is generally considered as a eukaryotic protein modification, which is catalysed by a three‐enzyme cascade and is reversed by deubiquitinating enzymes. Ubiquitination directs protein degradation and regulates cell signalling, thereby plays key roles in many cellular processes including immune response, vesicle trafficking and cell cycle. Bacterial pathogens inject a series of virulent proteins, named effectors, into the host cells. Increasing evidence suggests that many effectors hijack the host ubiquitin pathways to benefit bacterial infection. This review summarizes the known functions and mechanisms of effectors from human bacterial pathogens including enteropathogenic Escherichia coli, Salmonella, Shigella, Chlamydia and Legionella, highlighting the diversity in their mechanisms for manipulating the host ubiquitin pathways. Many effectors adopt the molecular mimicry strategy to harbour similar structures or functional motifs with those of the host E3 ligases and deubiquitinases. On the other hand, a few of effectors evolve novel structures or new enzymatic activities to modulate various steps of the host ubiquitin pathways. The diversity in the mechanisms enhances the efficient exploitation of the host ubiquitination signalling by bacteria.     

Acid stress damage of DNA is prevented by Dps binding in <Emphasis Type="Italic">Escherichia coli</Emphasis>O157:H7

Background  

Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Mass spectrometry-based quantitative proteomic analysis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis protein expression upon exposure to hydrogen peroxide

Background  

Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H₂O₂), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H₂O₂ or to host cells in vivo during the established phase of systemic infection have not been extensively studied.     

In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

Background  

In silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing). The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions.     

Histone H3K14 and H4K8 hyperacetylation is associated with Escherichia coli-induced mastitis in mice

Mastitis is a multietiological complex disease, defined as inflammation of parenchyma of mammary glands. Bacterial infection is the predominant cause of mastitis, though fungal, viral and mycoplasma infections also have been reported. Based on the severity of the disease, mastitis can be classified into subclinical, clinical and chronic forms. Bacterial pathogens from fresh cow milk were isolated and classified by standard microbiological tests and multiplex PCR. Epidemiological studies have shown that Escherichia coli is the second largest mastitis pathogen after Staphylococcus aureus in India. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profile and presence of virulence genes, a field isolate of E. coli was used for intramammary inoculation in lactating mice. Histopathological examination of hematoxylin and eosin stained sections showed severe infiltration of polymorphonuclear neutrophils, mononuclear inflammatory cells in the alveolar lumen and also in interstitial space, and necrosis of alveolar epithelial cells after 24 h. Western blot and immunohistochemical analysis of mice mammary tissues showed significant hyperacetylation at histone H3K14 residue of both mammary epithelial cells and migrated inflammatory cells. Quantitative real-time PCR and genome-wide gene expression profile in E. coli infected mice mammary tissue revealed differential expression of genes related to inflammation, immunity, antimicrobial peptide expression, acute phase response and oxidative stress response. Expression of milk proteins was also suppressed. ChIP assay from paraffinized tissues showed selective enrichment of acetylated histone H3K14 and H4K8 at the promoters of overexpressed genes. These data suggest that E. coli infection in mice mammary tissue leads to histone hyperacetylation at the promoter of immune genes, which is a pre-requisite for the expression of inflammatory genes in order to mount a drastic immune response.