Genetic basis of virulence in Shigella species.

Shigella species and enteroinvasive strains of Escherichia coli cause disease by invasion of the colonic epithelium, and this invasive phenotype is mediated by genes carried on 180- to 240-kb plasmids. In addition, at least eight loci on the Shigella chromosome are necessary for full expression of virulence. The products of these genes can be classified as (i) virulence determinants that directly affect the ability of shigellae to survive in the intestinal tissues, e.g., the aerobactin siderophore (iucABCD and iutA), superoxide dismutase (sodB), and somatic antigen expression (rfa and rfb); (ii) cytotoxins that contribute to the severity of disease, e.g., the Shiga toxin (stx) and a putative analog of this toxin (flu); and (iii) regulatory loci that affect the expression of plasmid genes, e.g., ompR-envZ, which mediates response to changes in osmolarity, virR (osmZ), which mediates response to changes in temperature, and kcpA, which affects the translation of the plasmid virG (icsA) gene which is associated with intracellular bacterial mobility and intracellular bacterial spread. A single plasmid regulatory gene (virF) controls a virulence-associated plasmid regulon including virG (icsA) and two invasion-related loci, i.e., (i) ipaABCD, encoding invasion plasmid antigens that may be structural components of the Shigella invasion determinant; and (ii) invAKJH (mxi), which is necessary for insertion of invasion plasmid antigens into the outer membrane.     

Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA , was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid-borne ipa operon. Expression of sipE and sipB in a Shigella non-invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.     

Identification of two targets of the type III protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella IpaD and IpaA proteins.

An important virulence factor of Salmonella spp. is their ability to gain access to host cells. A type III secretion system encoded in the inv and spa loci of these organisms is essential for this phenotype. We have identified two proteins, SipA and SipD, whose secretion from the bacterial cells is dependent on this system. The genes encoding these proteins are located at centisome 63 on the S. typhimurium chromosome, immediately downstream of the previously identified sipB and sipC genes (K. Kaniga, S. Tucker, D. Trollinger, and J. E. Galán, J. Bacteriol. 177:3965-3971, 1995). Nucleotide sequence analysis of the genes encoding these proteins indicated that SipA and SipD have significant sequence similarity to the Shigella IpaA and IpaD proteins. A nonpolar null mutation in sipD rendered S. typhimurium severely deficient for entry into cultured epithelial cells. In addition, this mutant strain exhibited increased secretion of a selected group of proteins whose export is controlled by the inv- and spa-encoded translocon. In contrast, a nonpolar mutation in sipA did not result in an invasion defect or in a significant decreased in virulence in a mouse model of infection. In addition, we have found an open reading frame immediately downstream of SipA that encodes a predicted protein with significant similarity to a family of acyl carrier proteins.     

Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length

The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 microm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.     

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.     

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.     

Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness.

The invasion of colonic epithelial cells by Shigella, an early essential step for causing bacillary dysentery, is mediated by the IpaB, IpaC and IpaD proteins. Secretion of the Ipa proteins from Shigella requires functions encoded by the mxi and spa loci. In this study, we show that contact between the bacteria and epithelial cell triggers release of the Ipa proteins into the external medium, which results in a rapid decrease in levels of Ipa proteins presented on the cell surface. When the bacteria were used to infect polarized Caco-2 cells, release of Ipa proteins occurred efficiently from bacteria interacting with the basolateral surface rather than with the apical surface. Moreover, the interaction of bacteria with components of the extracellular matrix, such as fibronectin, laminin or collagen type IV, also stimulates the release of Ipa proteins. The release of Ipa proteins from Shigella required the surface-located Spa32 protein encoded by one of the spa genes on the large plasmid.     

A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.     

Isolation of Shigella dysenteriae type 1 and S. flexneri strains from surface waters in Bangladesh: comparative molecular analysis of environmental Shigella isolates versus clinical strains

Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.     

Molecular basis of Salmonella-induced enteritis

Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non-typhoid Salmonella spp. The Salmonella pathogenicity island-1-encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.     

Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the ipaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 NotI restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence-associated chromosomal loci.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

Developing live Shigella vaccines using lambda Red recombineering

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.     

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.     

Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

Salmonella infection in its mammalian host can be dissected into two main components. The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand. The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently. This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention. Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions. Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response. Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance. Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.     

Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.