Phylogenetic analyses of the constituents of Type III protein secretion systems.

Multicomponent Type III protein secretion systems transfer gram-negative bacterial virulence factors directly from the bacterial cytoplasm to the cytoplasm of a host eukaryotic cell in a process that may involve a single energy-coupled step. Extensive evidence supports the conclusion that the genetic apparatuses that encode these systems have been acquired independently by different gram-negative bacteria, presumably by lateral transfer. In this paper we conduct phylogenetic analyses of currently sequenced constituents of these systems and their homologues. The results reveal the relative relatedness of these systems and show that they evolved with little or no exchange of constituents between systems. This fact suggests that horizontal transmission of the genes encoding these systems always occurred as a unit without the formation of hybrid gene clusters. Moreover, homologous flagellar proteins show phylogenetic clustering that suggests that the flagellar systems and Type III protein secretory systems diverged from each other following very early duplication of a gene cluster sharing many (but not all) genes. Phylogenies of most or all of the flagellar proteins follow those of the source organisms with little or no lateral gene transfer suggesting that homologous flagellar proteins are true orthologues. We suggest that the flagellar apparatus was the evolutionary precursor of Type III protein secretion systems.

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens.

Live attenuated Salmonella spp. are promising candidates as oral vaccine delivery systems for heterologous antigens. Clinical trials have demonstrated that this approach is feasible for human vaccinations but further optimisation is necessary to obtain a better efficacy. Here, we discuss how existing clinical and pre-clinical data can be used to guide such optimisation efforts.

Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi.

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.

Type III protein secretion systems in bacterial pathogens of animals and plants.

Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1.

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717-724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily.

The Salmonella typhimurium PhoP-repressed locus prgHIJK encodes components of a sec-independent type III secretion apparatus. This apparatus is composed of at least 17 proteins encoded on a 40 kb pathogenicity Island located at centisome 63 on the S. typhimurium chromosome. The secretion apparatus and some of its targets, SapB, SapC and SspD, are necessary for epithelial cell invasion. The transcription of many invasion genes, including prgHIJK, is coordinately activated by HilA, a transcription factor encoded within the pathogenicity island. In this report we identify sirA, a gene located outside the pathogenicity island that is essential for induction of prgHIJK and hilA transcription. sirA encodes a 234-amino-acid protein that is essential for S. typhimurium Ssp (Salmonella secreted protein) secretion and invasion and is similar to response regulators of two-component regulatory systems. sirA-mutant phenotypes could be suppressed by two DNA clones from unlinked loci, designated sirB and sirC. These data suggest that SirA may be phosphorylated in response to S. typhimurium sensing a mammalian microenvironment. Furthermore, SirA phosphorylation is predicted to initiate a cascade of transcription-factor synthesis which results in invasion-gene transcription, Ssp secretion, and bacterial invasion of epithelia.

Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes (SspB, sspC, sspD, and sspA), located between spaT and prgH, which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins IpaB, IpaC, IpaD and IpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87, 42 and 36 kDa. Complementation and analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA. sspC and sspD, but not sspA, are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK, homologous to the Shigella Ipa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63, 59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri Ipa proteins.

Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium.

A mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3'-terminal region of ccpA, where an in-frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C-terminus of CcpA. An in-frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single-copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid-encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a single-copy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis-active catabolite-responsive element to exert catabolite repression.

Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria?

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenolpyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fructose-1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.

Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria.

One form of catabolite repression (CR) in the Gram-positive genus, Bacillus, is mediated by a cis-acting element (CRE). We use here a consensus sequence to identify such elements in sequenced genes of Gram-positive bacteria. These are analysed with respect to position and type of gene in which they occur. CRE sequences near the promoter region are mainly identified in genes encoding carbon catabolic enzymes, which are thus likely to be subject to CR by a global mechanism. Functional aspects of CREs are evaluated.

Sequences of ccpA and two downstream Bacillus megaterium genes with homology to the motAB operon from Bacillus subtilis.

A regulatory gene with 69% nucleotide sequence identity to the Bacillus subtilis ccpA was cloned from Bacillus megaterium by complementation of a mutant relieved of catabolite repression. Sequencing of the gene and its adjacent regions revealed two additional open reading frames (ORFs) downstream from ccpA. These three genes are presumably in one operon. ORF1 and ORF2 show homology to two genes downstream from ccpA in B. subtilis, as well as to the B. subtilis motA and motB genes, respectively.

Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of an unrelated sequence, and glucose exerts additional xylR-dependent repression.

Catabolite repression (CR) of xylose utilization by Bacillus subtilis involves a 14-bp cis-acting element (CRE) located in the translated region of the gene encoding xylose isomerase (xylA). Mutations of CRE making it more similar to a previously proposed consensus element lead to increased CR exerted by glucose, fructose, and glycerol. Fusion of CRE to an unrelated, constitutive promoter confers CR to beta-galactosidase expression directed by that promoter. This result demonstrates that CRE can function independently of sequence context and suggests that it is indeed a generally active cis element for CR. In contrast to the other carbon sources studied here, glucose leads to an additional repression of xylA expression, which is independent of CRE and is not found when CRE is fused to the unrelated promoter. This repression requires a functional xylR encoding Xyl repressor and is dependent on the concentrations of glucose and the inducer xylose in the culture broth. Potential mechanisms for this glucose-specific repression are discussed.