A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis.

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to 'anti-PG' antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the 'chlamydial anomaly'). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

Virulence protein export systems in Salmonella and Shigella: a new family or lost relatives?

A number of Gram-negative bacterial pathogens secrete 'virulence determinants' directly into the extracellular medium, where they interact with host cells to promote disease. The study of the secretion machinery used by these organisms to transport specific virulence determinants out to the cell surface and beyond is of growing importance in the field of bacterial pathogenesis. Elements of the secretion machinery are shared by several pathogens. These homologous elements may lead to a better understanding of how the machinery works, but the unique elements will tell us more about what distinguishes one bacterial pathogen from another.

Prevalence of Shiga toxin-producing Shigella species isolated from French travellers returning from the Caribbean: an emerging pathogen with international implications.

Shiga toxins (Stxs) are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Stx-producing Escherichia coli or Shigella dysenteriae serotype 1, and infections with these strains can lead to haemolytic-uraemic syndrome. Over the past decade, there has been increasing recognition that Stx is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage, and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study, we further explored the epidemiological link to this region by utilizing the French National Reference Centre for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travellers returning from the Caribbean. Approximately 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travellers who were infected with Stx-producing Shigella had recently travelled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing showed that the toxin genes were encoded by a prophage that was highly identical to the phage that we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups.

Use of UV-irradiated bacteriophage T6 to kill extracellular bacteria in tissue culture infectivity assays.

We have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique we describe has the following characteristics: (a) bacterial killing is complete within 15 min at 37 degrees C, with a greater than 10(3)-fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues.

Regulation of virulence genes in Shigella.

Shigella pathogenicity is a multi-genic phenomenon involving the participation of genes on both the 230 kilobase virulence plasmid and the chromosome. A key feature of the regulation of Shigella virulence is its response to growth temperature. Genes in the virulence regulon are fully expressed at 37 degrees C, the normal temperature of Shigella's mammalian host, and the regulon is repressed at lower temperatures. Virulence gene expression is regulated in both a positive and a negative fashion by several plasmid-encoded activators and at least one chromosomally encoded repressor. The use of a variety of molecular tools including gene fusions, cloning, complementation, DNA sequencing and mRNA analysis, has provided a more complete understanding of how various, unlinked genetic loci contribute in a co-ordinated fashion to the pathogenic phenotype expressed by Shigella.

Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence.

Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 37 degrees C but phenotypically avirulent (noninvasive) at 30 degrees C. A vir::lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac+ phenotype at the nonpermissive temperature. A transposon Tn10-induced mutant that was Lac+ at 30 degrees C and 37 degrees C was isolated, and the Tn10 insertion was transduced into a wild-type strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 30 degrees C and 37 degrees C. Other virulence-associated phenotypes were also deregulated and expressed at 30 degrees C. Southern hybridization with a probe for Tn10 determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and trp genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the Tn10-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of Tn10 into this gene inactivates a repressor that normally blocks expression of vir genes at 30 degrees C.

Identification of Shigella invasion genes by isolation of temperature-regulated inv::lacZ operon fusions.

Penetration and multiplication within cells of the human colonic epithelium are hallmarks of Shigella spp. pathogenicity. Shigella spp. virulence is regulated by growth temperature. Strains phenotypically virulent when grown at 37 degrees C are phenotypically avirulent when grown at 30 degrees C. The number of genes involved in Shigella spp. pathogenicity and how many virulence genes are temperature regulated are unknown. To facilitate the study of temperature-regulated virulence in Shigella spp., we employed lacZ operon fusion technology to identify temperature-regulated invasion (inv) genes. Four inv::lacZ fusion mutants were identified and found to be unable to invade HeLa cells. The fusions were located in a region of the 220-kilobase invasion plasmid defined as the minimal amount of DNA required for invasion, and they were controlled by virR, the temperature-dependent virulence gene regulator. Western blot (immunoblot) and Southern hybridization analyses indicated that one of the fusions was located in a known inv gene, ipaB, which encodes one of the major immunogenic peptides of Shigella spp. This ipaB::lacZ operon fusion mutant synthesized a truncated IpaB protein recognized by IpaB-specific monoclonal antibodies. Three of the fusions were within novel genes mapping to regions previously identified as essential for a positive virulence phenotype. Analysis of bacterial surface proteins suggested that the genes marked by these fusions may play a role in the correct surface expression of the ipaB and ipaC gene products.

Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence.

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.

mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis.

The plasmid-encoded invasion plasmid antigen (Ipa) export accessory locus of Shigella flexneri 2a, mxiA, was cloned, and the complete DNA sequence of the gene was determined. The mixA open reading frame was found to encode a polypeptide of 74.03 kDa with a pI of 5.02. A hydropathy analysis of the predicted protein revealed a hydrophilic C terminus and an extremely hydrophobic N terminus without a cleavable signal sequence but with several potential membrane-spanning regions. While a homology search did not reveal any significant relatedness of the mxiA DNA sequence to any known bacterial gene sequences, the derived amino acid sequence of MxiA was found to be highly homologous (68%) to the sequence of the protein encoded by the low-calcium-response locus, lcrD, of Yersinia pestis. The lcrD encodes an inner membrane regulatory protein that has an N-terminal membrane anchor and that is implicated in facilitating the export of Y. pestis outer membrane proteins (G. V. Plano, S. S. Barve, and S. C. Straley, J. Bacteriol. 173:7293-7303, 1991). Congo red binding, HeLa cell invasion, and Ipa excretion were restored in two avirulent mxiA fusion mutants when they were transformed with a cloned copy of the mxiA gene. Furthermore, the expression of the cloned mxiA gene was independent of any vector-specified promoter, suggesting that the transcription of mxiA is driven by its own promoter in this clone. In contrast, the overexpression of mxiA that resulted when it was placed under the control of the lac promoter was found to be deleterious in Escherichia coli. We conclude that mxiA is a homolog of the Y. pestis lcrD locus and may function similarly in S. flexneri, either by directly affecting the excretion of virulence factors or by regulating the expression of export accessory genes.

Spa33, a cell surface-associated subunit of the Mxi-Spa type III secretory pathway of Shigella flexneri, regulates Ipa protein traffic.

The Mxi-Spa type III secretion system of Shigella flexneri directs the host cell contact-induced secretion of a set of invasins, referred to as Ipas. In this study, we examined the role of Spa33 in Ipa secretion. A spa33-null mutant was both noninvasive and unable to translocate the Ipas from inner membrane to outer membrane (OM) positions of the Mxi-Spa transmembrane channel. Spa33 was found to be a Mxi-Spa substrate that is translocated to the bacterial cell surface upon the induction of Ipa secretion. This mobility may serve to drive Ipa translocation within Mxi-Spa toward OM positions. Consistent with a second distinct role in regulating Ipa traffic, the overexpression of Spa33 also blocked Ipa secretion and resulted in Ipa accumulation at the OM. Co-overexpression of Spa33 and another OM-associated element, Spa32, did not disrupt Ipa secretion, suggesting an interaction between the two proteins and an effect on the mechanism which serves to regulate Ipa release from the OM. These findings indicate that Spa33 is a mobile element within Mxi-Spa, which is required to control Ipa translocation into and out of OM positions of the secretory structure.

Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence.

Quorum-sensing systems regulate the expression of virulence factors in a wide variety of plant and animal pathogens, including members of the Enterobacteriaceae. Studies of Shigella virulence gene expression have demonstrated that maximal expression of genes encoding the type III secretion system and its substrates and maximal activity of this virulence organelle occur at high cell density. In these studies, we demonstrate that the expression of ipa, mxi, and spa invasion operons is maximal in stationary-phase bacteria and that conditioned media derived from stationary-phase cultures enhance the expression of these loci. In contrast, expression of virB, a transcription factor essential for the expression of invasion loci, peaks in late log phase; accordingly, virB expression is enhanced by a signal(s) present in conditioned media derived from late-log-phase cultures. Autoinducer 2 (AI-2), a quorum signaling molecule active in late log phase, was synthesized by Shigella species and enteroinvasive Escherichia coli and shown to be responsible for the observed peak of virB expression. However, AI-2 does not influence invasion operon expression and is not required for Shigella virulence, as mutants deficient in AI-2 synthesis are fully virulent. The implications of these findings with regard to both virB and invasion operon expression and the evolution of circuitries governing virulence gene expression are discussed.

MxiM and MxiJ, base elements of the Mxi-Spa type III secretion system of Shigella, interact with and stabilize the MxiD secretin in the cell envelope.

The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in Shigella) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element in Shigella, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.

Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression.

Expression of the predominantly plasmid-encoded virulence regulon of Shigella flexneri 2a is induced by growth at 37 degrees C and repressed by growth at 30 degrees C. During growth at 37 degrees C, spontaneous S. flexneri mutants arise which have undergone virulence plasmid curing or rearrangement and no longer display the virulent phenotype. In the laboratory, the unstable nature of the virulence plasmid causes complete loss of virulence in a growing population. We have undertaken an analysis of virulence plasmid instability, classifying events which produced individual avirulent derivatives within a virulent population and identifying the factor(s) which controlled conversion. Multiplex PCR analysis of DNA obtained from spontaneous avirulent derivatives indicated that virF and virB were deleted or otherwise inactivated in over 97% of the isolates. The virF and virB loci encode regulatory proteins required for transcriptional activation of the virulence regulon. Inactivation of these key regulatory loci in the vast majority of avirulent derivatives which arose during growth at 37 degrees C suggested that virulence gene expression induced virulence plasmid instability. Consistent with this hypothesis, we observed stable virulence plasmid maintenance during growth of a wild-type strain at 30 degrees C where virulence gene expression was repressed. The virulence plasmid was also stably maintained in virF and virB mutants grown at 37 degrees C. Conversely, virulence plasmid destabilization was induced at 30 degrees C and accelerated at 37 degrees C through expression of VirF or VirB from multicopy plasmids. These results indicate that exposure of S. flexneri to conditions favoring induction of the virulent phenotype also favor its loss. The significance of this paradox of Shigella pathogenicity is discussed.

Bacteriophage Mu d1(Apr lac) generates vir-lac operon fusions in Shigella flexneri 2a.

Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes, we set out to develop a rapid, easily-assayed phenotype with which to measure expression of virulence. This report described a procedure for isolating vir-lac operon fusions in S. flexneri 2a by using the specialized transducing bacteriophage Mu d1(Apr lac) of Casadaban and Cohen (M. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1976). Mu d1(Apr lac) lysogens were isolated and screened for loss of virulence and for temperature-dependent expression of the lactose genes on Mu d1(Apr lac). A recombinant plasmid carrying the Mu immunity gene was also introduced into lysogens of interest to stabilize the Mu d1(Apr lac) insertion and prevent possible thermal induction at 37 degrees C. The mutant which we isolated failed to penetrate tissue culture cells in the assay for virulence and produced almost 15-fold more beta-galactosidase when grown at 37 degrees C than when grown at 30 degrees C. The site of insertion of Mu d1(Apr lac) in this strain was shown to be in the 140-megadalton plasmid pSf2a140, which is known to be associated with virulence. P1L4-mediated transduction of the insertion into a virulent recipient demonstrated genetic linkage of Mu d1(Apr lac) with loss of virulence and temperature-dependent expression of beta-galactosidase. All of these features fulfill the phenotype expected for a Mu d1(Apr lac)-induced vir-lac operon fusion. This mutant provides us with a means of measuring expression of a gene function required for virulence by assaying for beta-galactosidase. The insertion will also serve as a starting point for mapping of genes on pSf2a140 which are necessary for expression of virulence.

The mxi-Spa type III secretory pathway of Shigella flexneri requires an outer membrane lipoprotein, MxiM, for invasin translocation.

Invasion of epithelial cells by Shigella flexneri is mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here that mxiM, part of the mxi-spa locus in the S. flexneri virulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in a mxiM null mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.

Establishment of unipolar localization of IcsA in Shigella flexneri 2a is not dependent on virulence plasmid determinants.

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.

Studies on the antigenic determinants of the Thy-1.2 alloantigen as expressed by the murine lymphoblastoid line S-49.1 TB-2-3.

Sialic acid has been shown to be part of the antigenic determinant of the Thy-1.2 alloantigen as expressed by the murine cell line S-49.1 TB-2-3 (S-49). This conclusion is based on the loss of cytotoxic inhibitory activity by the action of neuraminidase on the Thy-1.2 alloantigen. Also, sialic acid has the ability to inhibit the cytotoxic assay of AKR anti-C3H Thy-1.2 serum for S-49 cells. Further evidence for the protein nature of the Thy-1.2 alloantigen is apparent by trypsin digestion. Possibly the Thy-1.2 alloantigen as expressed on S-49 cells is a glycoprotein.

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)).

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.

Pathoadaptive mutations that enhance virulence: genetic organization of the cadA regions of Shigella spp.

Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in Shigella species that have evolved from LDC-expressing Escherichia coli. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of Shigella enterotoxins and that the gene encoding LDC, cadA, was abolished by large chromosomal deletions in each Shigella species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of the cad region were sequenced from the four Shigella species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral E. coli chromosome displaced the cadA locus to form distinct genetic linkages that are unique to each Shigella species. Hybridization studies, using an E. coli K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the Shigella genomes. None of these novel gene arrangements were observed in representatives of all E. coli phylogenies. Collectively, these observations indicate that inactivation of the cadA antivirulence gene occurred independently in each Shigella species. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal E. coli, strong pressures in host tissues selected Shigella clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.