N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.     

Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrE_Lm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrE_Lm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrE_Lm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.     

Identification of genes involved in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm formation by <Emphasis Type="Italic">mariner</Emphasis>-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.     

Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo

Background

Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species.

Methods and Findings

We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models.Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection.

Conclusion

This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.     

Identification and Characterization of Di- and Tripeptide Transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[¹⁴C]Ala was mediated by the DtpT transporter and was abrogated in a ΔdtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the ΔdtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.     

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20–30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. An L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.     

Identification of a new molecular target of class IIa bacteriocins in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> EGDe

It was shown recently (Calvez et al. 2007) that glpQ and pde genes are involved in intermediate resistance of Enterococcus faecalis JH2-2 to DvnV41, a class IIa bacteriocin produced by Carnobacterium divergens V41. The listerial orthologs of lpQ and pde genes might be lmo0052 and lmo1292 genes, respectively. Here, the role of lmo0052 and lmo1292 genes in resistance of Listeria monocytogenes EGDe to DvnV41 and MesY105 was investigated. L. monocytogenes EGDe was inactivated in lmo0052 and/or lmo1292 by homologous recombination. Listerial mutant strain EGDSC02 (inactivated in the putative glpQ gene), was slightly resistant to DvnV41. The listerial mutant strain EGDSC01 (inactivated in the putative pde gene) remained, as the wild-type strain, sensitive to DvnV41, but was affected in growth parameters.     

Identification and Disruption of BetL, a Secondary Glycine Betaine Transport System Linked to the Salt Tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N,N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus ς^B-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed K_m value of 7.9 μM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL⁺ parent, signifying the role of the transporter in Listeria osmotolerance.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Contribution of Chitinases to Listeria monocytogenes Pathogenesis

Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis.Listeria monocytogenes is a Gram-positive bacterium that is found as an inhabitant of soil, water, and decaying vegetation, where the organism is believed to live as a saprophyte (12, 13, 16). Upon consumption of the bacterium by a susceptible host, L. monocytogenes adopts a pathogenic lifestyle that can lead to serious and sometimes fatal infections (1, 3, 15, 22, 32). L. monocytogenes is considered a major food-borne pathogen and has been responsible for some of the largest and most expensive food recalls in U.S. history (6-10, 27, 31). While significant attention has focused on the identification of L. monocytogenes gene products that specifically contribute to bacterial life within host cells, relatively less is known regarding the function of gene products that may be adapted to contribute to bacterial life both inside and outside the infected host.Chitinases catalyze the hydrolysis of chitin, a linear polymer of N-acetylglucosamine residues linked in β-1,4 glycosidic bonds (5). Microbial chitinases have been associated with nutrient acquisition, as chitin is abundant in the environment and can serve as a source of carbon and nitrogen (17). Recently, a chitinase and a chitin binding protein have been linked to bacterial virulence for two environmental pathogens, Legionella pneumophila (14) and Vibrio cholerae (19, 21). Although chitin is not synthesized by mammals, L. pneumophila chitinase has been implicated in enhancing bacterial colonization of the lungs of infected mice (14), while a chitin binding protein produced by V. cholerae has been implicated in intestinal colonization (19, 21).L. monocytogenes produces two chitinases encoded by lmo1883 (chiA) and lmo0105 (chiB) and one chitin binding protein encoded by lmo2467 (Fig. ​(Fig.11 A) (26). chiA and chiB encode proteins containing a glycosyl hydrolase family 18 chitinase domain, and both have been demonstrated to possess chitinolytic activity (25, 26). Interestingly, the expression of chiA has recently been reported to be induced within the cytosol of infected macrophages (11). lmo2467 encodes a hypothetical chitin binding protein that lacks a chitinase domain (26). To investigate whether chiA, chiB, or lmo2467 contributes to host infection, in-frame deletion mutants were constructed in each gene in the L. monocytogenes chromosome using allelic exchange (18). Each of the resulting in-frame deletion mutants was found to resemble wild-type L. monocytogenes with respect to bacterial growth in brain heart infusion (BHI) broth culture (S. Chaudhuri and N. Freitag, data not shown), and the deletion of either chiA or chiB significantly reduced secreted chitinase activity as detected in the culture supernatants of mutant strains following 48 h of growth in LB at 30°C (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.L. monocytogenes genes encoding chitinases and chitin binding proteins. (A) chiA (lmo1883) encodes a chitinase of 352 amino acids, chiB (lmo0105) encodes a chitinase of 756 amino acids, and lmo2467 encodes a predicted chitin binding protein of 478 amino acids (26). Chromosomal deletion mutants were constructed for each gene indicated in black. Surrounding coding regions are represented by white arrows. (B) Chitinase activity detected in supernatants derived from wild-type, ΔchiA, ΔchiB, and Δlmo2467 strains. Bacterial cultures were grown for 48 h at 30°C in LB broth. Bacterial supernatants were assayed for secreted chitinase activity using the substrate 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (14, 25). Chitinase units are expressed as the absorbance at 405 nm times 100 divided by the culture optical density measured at 600 nm (OD₆₀₀). Data shown are representative of two independent experiments.The ability of L. monocytogenes chitinase and chitin binding protein mutants to invade host cells, escape from the phagosome, and replicate in the cytosol was investigated via bacterial infection of the human intestinal Caco-2 cell line (Fig. ​(Fig.22 A). Monolayers of Caco-2 cells were grown on glass coverslips and infected with L. monocytogenes at a multiplicity of infection (MOI) of 10 bacteria per cell. At 1 h postinfection, monolayers were washed and medium containing 10 μg/ml gentamicin was added to kill extracellular bacteria. Intracellular bacterial replication was monitored by removing the coverslips at the indicated time points postinfection, lysing the infected host cells by vortexing the coverslips in water, and plating for viable CFU on LB agar media (29). Bacterial invasion and intracellular growth of each mutant strain in Caco-2 cells were found to be similar to those of wild-type L. monocytogenes (Fig. ​(Fig.2A).2A). Identical patterns of bacterial replication were also observed in the murine-derived J774 macrophage-like cell line using a similar approach (S. Chaudhuri and N. Freitag, data not shown).Open in a separate windowFIG. 2.Chitinase and chitin binding protein deletion mutants exhibit normal patterns of intracellular growth and cell-to-cell spread in tissue culture cells. (A) Monolayers of Caco-2 intestinal epithelial cells grown on glass coverslips were infected with L. monocytogenes wild-type (•), ΔchiA (▪), ΔchiB (▴), and Δlmo2467 (▾) strains at an MOI of 10:1. After 1 h, monolayers were washed with phosphate-buffered saline (PBS) and gentamicin was added to kill any remaining extracellular bacteria. Coverslips were removed at the indicated time points, followed by host cell lysis and enumeration of bacterial CFU on agar media. Data are representative of three independent experiments. (B) Monolayers of L2 fibroblast cells were infected with wild-type, ΔchiA, ΔchiB, and Δlmo2467 bacteria as previously described (30). Bacterial intracellular growth and cell-to-cell spread were monitored by measuring the diameter of the zones of clearance (plaques) in the infected monolayers. Relative plaque diameters are indicated as a percentage of those of the wild type (WT), which was set to 100%. Data shown are the average plaque diameter ± standard error from results from three independent experiments.The ability of L. monocytogenes to spread to adjacent cells during tissue culture infection can be rapidly assessed by measuring the ability of bacteria to form small zones of cell clearing or plaques during the infection of mouse fibroblast cell monolayers (30). Mouse L2 fibroblast cells were grown in 3.5-cm wells and infected with L. monocytogenes as previously described (30). One hour postinfection, the monolayers were washed and gentamicin was added along with a Dulbecco modified Eagle medium (DMEM)-0.7% agar overlay. Plaque formation was assessed at 3 days postinfection by staining viable L2 cells with neutral red. The chitinase and chitin binding protein mutants formed plaques in L2 cell monolayers with the same efficiency and of the same diameter as those formed by wild-type L. monocytogenes (Fig. ​(Fig.2B),2B), indicating that each of the mutant strains was fully capable of intracellular bacterial replication and cell-to-cell spread.The chitinase and chitin binding protein mutants were then tested for virulence in a mouse model of infection. ND4 Swiss Webster mice were infected via tail vein injection (2) with the ΔchiA and ΔchiB chitinase deletion mutants as well as with the Δlmo2467 mutant and wild-type L. monocytogenes (Fig. ​(Fig.3).3). Each mouse was inoculated with 2 × 10⁴ CFU, and at 3 days postinfection, the animals were sacrificed and the numbers of bacterial CFU were determined from homogenates of isolated livers and spleens. Each of the mutants exhibited a significant defect in bacterial replication within infected mice (Fig. ​(Fig.3).3). The Δlmo2467 mutant exhibited the most modest level of attenuation, with approximately 4-fold fewer bacteria recovered from the livers and 6-fold fewer from the spleens of infected animals than from those infected with wild-type L. monocytogenes (Fig. ​(Fig.3).3). The ΔchiB mutant exhibited more significant levels of attenuation, with approximately 8-fold and 14-fold fewer bacteria recovered from the livers and spleens, respectively (Fig. ​(Fig.3).3). Attempts to complement the defect of the ΔchiB mutant by providing a wild-type copy of the gene in trans using plasmid vector pPL2 (24) were not successful, as it was not possible to recover stable L. monocytogenes plasmid integrants for reasons that are not apparent (S. Chaudhuri and N. Freitag, data not shown). The most dramatic virulence defect was observed for strains lacking chiA (Fig. ​(Fig.3).3). The absence of chiA reduced bacterial colonization of the liver and spleen more than 19-fold and 45-fold, respectively, and the defect could be fully complemented in the liver and almost fully complemented in the spleen by providing a PCR-amplified wild-type copy of chiA in trans on the integrative plasmid vector pPL2 (24). These data indicate a significant role for the chitinase ChiA in L. monocytogenes virulence and suggest that additional contributions are made by chiB and lmo2467 during mouse infection.Open in a separate windowFIG. 3.L. monocytogenes chitinase mutants are defective for growth in the livers and spleens of infected mice. ND4 Swiss Webster mice were infected via tail injection (2) with 2 × 10⁴ CFU of wild-type, ΔchiA, ΔchiB, Δlmo2467, and ΔchiA bacteria complemented with plasmid-borne chiA (ΔchiA+pPL2-chiA). After 72 h of infection, the animals were euthanized and the bacterial burdens were measured in the livers (A) and spleens (B). A minimum of 5 mice were inoculated per strain tested, and the mean and standard deviation are shown. Statistical significance was determined using one-way analysis of variance with Tukey''s multiple-comparison test (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).Given the absence of chitin in mammals, what are the potential roles for chitinases and chitin binding proteins in L. monocytogenes virulence? For the secreted V. cholerae chitin binding protein GbpA, it has been reported that the protein serves to enhance bacterial colonization of the intestine through the binding of mucin (4, 21), which consists of glycoproteins rich in carbohydrates that include N-acetylglucosamine (28). Kawada et al. have recently reported that a chitin binding protein expressed by Serratia marcescens increased bacterial adhesion to colonic epithelial cells via binding to the chitinase 3-like 1 (CHI3L1) protein expressed on the host cell surface (20). The L. pneumophila chitinase has been reported to contribute to bacterial colonization of mouse lung; however, the mechanism by which the enzyme enhances bacterial colonization of lung epithelium has not yet been established (14). For L. monocytogenes, neither ChiA, ChiB, nor Lmo2467 appeared to have any influence on bacterial invasion or replication within tissue culture cell lines (Fig. ​(Fig.2),2), although the proteins clearly contributed to virulence via bloodstream infection of mice (Fig. ​(Fig.3).3). It remains possible that one or more of the secreted proteins recognize glycoproteins or carbohydrate moieties present on host cells (but not expressed by the tissue culture cell lines examined in this study) and that this recognition somehow serves to enhance bacterial uptake. Alternatively, the expression of chiA has been reported to be induced within infected macrophages (11) and also by the L. monocytogenes central virulence regulator PrfA (23), raising the possibility that ChiA and/or the other chitin binding proteins may serve to modify host immune responses by binding to glycoproteins or other carbohydrate moieties that contribute to immune signaling. Elucidation of the role of chitinases and chitin binding proteins in L. monocytogenes pathogenesis should provide insight into how bacterial factors that contribute to survival in the outside environment can be exploited for additional roles within an infected mammalian host.     

Cell Wall Teichoic Acid Glycosylation in Listeria monocytogenes Serotype 4b Requires gtcA, a Novel, Serogroup-Specific Gene

We have identified a novel gene, gtcA, involved in the decoration of cell wall teichoic acid of Listeria monocytogenes serotype 4b with galactose and glucose. Insertional inactivation of gtcA brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid. Interestingly, the composition of membrane-associated lipoteichoic acid was not affected. Complementation of the mutants with the cloned gtcA in trans restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within L. monocytogenes, sequences homologous to gtcA were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b, gtcA appears to be the first member of a bicistronic operon which includes a gene with homology to Bacillus subtilis rpmE, encoding ribosomal protein L31. In contrast to gtcA, the latter gene appears conserved among all screened serotypes of L. monocytogenes.