Biomedical subjects
Publications and source records attributed to E A Groisman.
In many pathogens, virulence can be conferred by a single region of the genome. In contrast, the facultative intracellular lifestyle of Salmonella demands a large number of genes distributed around the chromosome. The evolution of Salmonella has been marked by the acquisition of several 'pathogenicity islands', each contributing to the unique virulence properties of this microorganism.
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The PmrA-PmrB two-component system of Salmonella typhimurium controls resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human neutrophils. Amino acid substitutions in the regulatory protein PmrA conferring resistance to polymyxin lower the overall negative charge of the lipopolysaccharide (LPS), which results in decreased bacterial binding to cationic polypeptides and increased bacterial survival within human neutrophils. We have now identified three PmrA-activated loci that are required for polymyxin resistance. These loci were previously shown to be necessary for growth on low-Mg2+ solid media, indicating that LPS modifications that mediate polymyxin resistance are responsible for the adaptation to Mg2+-limited environments. Conditions that promote transcription of PmrA-activated genes--growth in mildly acidic pH and micromolar Mg2+ concentrations--increased survival in the presence of polymyxin over 16,000-fold in a wild-type organism but not in a mutant lacking pmrA. Our experiments suggest that low pH and low Mg2+ concentrations may induce expression of PmrA-activated genes within phagocytic cells and promote bacterial resistance to host antimicrobial proteins. We propose that the LPS is a Mg2+ reservoir and that the PmrA-controlled LPS modifications neutralize surface negative charges when Mg2+ is transported into the cytoplasm during growth in Mg2+-limited environments.
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We have identified a region unique to the Salmonella typhimurium chromosome that is essential for virulence in mice. This region harbors at least three genes: two (spiA and spiB) encode products that are similar to proteins found in type III secretion systems, and a third (spiR) encodes a putative regulator. A strain with a mutation in spiA was unable to survive within macrophages but displayed wild-type levels of epithelial cell invasion. The culture supernatants of the spi mutants lacked a modified form of flagellin, which was present in the supernatant of the wild-type strain. This suggests that the Spi secretory apparatus exports a protease, or a protein that can alter the activity of a secreted protease. The "pathogenicity island" harboring the spi genes may encode the virulence determinants that set Salmonella apart from other enteric pathogens.
Ions are not traditionally thought to act as first messengers in signal transduction cascades. However, while searching for genes regulated by the PhoP/PhoQ virulence regulatory system of Salmonella typhimurium, we recovered two loci whose expression is controlled by the concentration of Mg2+. To determine whether Mg2+ is the signal modulating the whole PhoP/PhoQ system, we evaluated the gene expression pattern of six PhoP-activated genes. Growth in physiological concentrations of divalent cations repressed transcription of PhoP-activated genes and rendered wild-type Salmonella phenotypically PhoP-. Mg2+ changed the conformation of the periplasmic domain of PhoQ, identifying this protein as a Mg2+ sensor. A mutation in the sensing domain of PhoQ altered the set point for Mg2+ and rendered Salmonella avirulent.
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Protamine is a polycationic peptide found in the nuclei of sperm of different animal species. While it has long been known to have antimicrobial properties, its mode of action has remained elusive. We have investigated the mechanism of action of protamine and established that this peptide exerts its antibacterial effect without causing cell lysis or permeabilization of the cytoplasmic membrane. Respiring cells were more susceptible than nonrespiring cells, and loss of viability could be prevented by incubation at low pH or the addition of respiratory poisons. This indicates that protamine activity is influenced by the electrical membrane potential (delta psi): increased killing occurs at higher delta psi values. Protamine caused inhibition of proline uptake, rapid efflux of proline from preloaded cells, and a reduction in the cellular ATP content. Furthermore, protamine-treated cells first lost the ability to accumulate leucine and then could not carry out protein synthesis. Cumulatively, our data indicate that protamine disrupts energy transduction and nutrient uptake functions, and suggest that the cytoplasmic membrane is the target of protamine action.
We investigated the phylogenetic distribution of the SPI-1 and SPI-2 pathogenicity islands in Salmonella spp. SPI-1 was present in representatives of all eight subspecific groups, but no SPI-2-hybridizing sequences were detected in group V (S. bongori). Our data suggest that SPI-2 was acquired by S. enterica after its split from S. bongori.
The PhoP-PhoQ two-component system is essential for virulence in Salmonella typhimurium. This system controls expression of some 40 different proteins, yet most PhoP-regulated genes remain unknown. To identify PhoP-regulated genes, we isolated a library of 50,000 independent lac gene transcriptional fusion strains and investigated whether production of beta-galactosidase was regulated by PhoP. We recovered 47 lac gene fusions that were activated and 7 that were repressed when PhoP was expressed. Analysis of 40 such fusions defined some 30 loci, including mgtA and mgtCB, which encode two of the three Mg2+ uptake systems of S. typhimurium; ugd, encoding UDP-glucose dehydrogenase; phoP, indicative that the phoPQ operon is autoregulated; and an open reading frame encoding a protein with sequence similarity to VanX, a dipeptidase required for resistance to vancomycin. Transcription of PhoP-activated genes was regulated by the levels of Mg2+ in a PhoP-dependent manner. Strains with mutations in phoP or phoQ were defective for growth in low-Mg2+ media. The mgtA and mgtCB mutants reached lower optical densities than the wild-type strain in low-Mg2+ liquid media but displayed normal growth on low-Mg2+ solid media. Six PhoP-activated genes were identified as essential to form colonies on low-Mg'+ solid media. Cumulatively, our experiments establish that the PhoP-PhoQ system governs the adaptation to magnesium-limiting environments.
The PhoP/PhoQ two-component system of Salmonella typhimurium governs transcription of some 25 loci in response to the extracellular concentration of Mg2+. We have now identified one of these loci as pmrCAB, which codes for a two-component system that mediates resistance to the antibiotic polymyxin B. Transcription of seven of 25 PhoP-activated loci was dependent on a functional PmrA protein, the response regulator of the PmrA/PmrB system. Expression of the PmrA-dependent loci was induced by either Mg2+ limitation or mild acidification, whereas transcription of a PmrA-independent gene was activated by Mg2+ limitation but not acid pH. Induction of PmrA-activated genes by Mg2 limitation required the PhoP and PhoQ proteins. In contrast, the acid-mediated activation of PmrA-regulated genes occurred in strains that were missing either one of these proteins. Transcriptional regulation by a cascade of two-component systems allows pathogenic bacteria to express their virulence determinants in response to a broader spectrum of environmental cues.
For 21 strains of Salmonella enterica, nucleotide sequences were obtained for three invasion genes, spaO, spaP, and spaQ, of the chromosomal inv/spa complex, the products of which form a protein export system required for entry of the bacteria into nonphagocytic host cells. These genes are present in all eight subspecies of the salmonellae, and homologues occur in a variety of other bacteria, including the enteric pathogens Shigella and Yersinia, in which they are plasmid borne. Evolutionary diversification of the invasion genes among the subspecies of S. enterica has been generally similar in pattern and average rate to that of housekeeping genes. However, the range of variation in evolutionary rate among the invasion genes is unusually large, and there is a relationship between the evolutionary rate and cellular location of the invasion proteins, possibly reflecting diversifying selection on exported proteins in adaptation to variable host factors in extracellular environments. The SpaO protein, which is hypervariable in S. enterica and exhibits only 24% sequence identity with its homologues in Shigella and Yersinia, is secreted. In contrast, the membrane-associated proteins SpaP, SpaQ, and InvA are weakly polymorphic and have > 60% sequence identity with the corresponding proteins of other enteric bacteria. Acquisition of the inv/spa genes may have been a key event in the evolution of the salmonellae as pathogens, following which the invention of flagellar phase shifting facilitated niche expansion to include warm-blooded vertebrates.
The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.
Despite differences in disease pathologies and host range, many enteric pathogens, including Salmonella and Shigella spp., utilize a remarkably similar machinery to secrete proteins that promote their entry into host cells. Analogous structures are required for the export of virulence proteins in other animal and plant pathogens. While the structure and organization of the gene complexes specifying these secretory pathways are broadly conserved, their phylogenetic distribution and genomic locations suggest that these sequences arose independently in divergent pathogens.
The ability of invading pathogens to proliferate within host tissues requires the capacity to resist the killing effects of a wide variety of host defense molecules. sap mutants of the facultative intracellular parasite Salmonella typhimurium exhibit hypersensitivity to antimicrobial peptides, cannot survive within macrophages in vitro and are attenuated for mouse virulence in vivo. We conducted a molecular genetic analysis of the sapG locus and showed that it encodes a product that is 99% identical to the NAD+ binding protein TrkA, a component of a low-affinity K+ uptake system in Escherichia coli. SapG exhibits similarity with other E. coli proteins implicated in K+ transport including KefC, a glutathione-regulated efflux protein, and Kch, a putative transporter similar to eukaryotic K+ channel proteins, sapG mutants were killed by the antimicrobial peptide protamine in the presence of both high and low K+, indicating that protamine hypersensitivity is not due to K+ starvation. Strains with mutations in sapG and either sapJ or the sapABCDF operon were as susceptible as sapG single mutants, suggesting that the proteins encoded by these loci participate in the same resistance pathway. SapG may modulate the activities of SapABCDF and SapJ to mediate the transport of peptides and potassium.
Since diverging from a common ancestor some 120 million years, Escherichia coli and Salmonella typhimurium have accumulated numerous phenotypic characteristics which have traditionally been used to distinguish these enteric species. While most of the genetic differences between these species are due to the accumulation of point mutations, the majority of the observed variation in phenotypic characters is attributable to segments of the genome confined to only one of the species. We have analyzed the map positions, G+C contents, nucleotide sequences and functions of regions unique to the Salmonella chromosome in an attempt to determine the ancestry of species-specific sequences. Some of the Salmonella-specific regions had uncharacteristically low base compositions and contained open reading frames of atypical codon usage patterns suggesting that portions of the genome were acquired by horizontal transfer from distantly-related bacterial species. The role of these species-specific sequences was assayed by constructing mutant strains harboring deletions in the corresponding regions of the genome. Several functions were ascribed to these unique portions of the Salmonella chromosome, including one encoding proteins involved in virulence and invasion of host epithelial cells.
For most bacterial species, virulence is viewed as a derived state, whereby pathogens acquire certain loci and are rendered virulent. The majority of virulence genes in Salmonella are present in closely related nonpathogenic species, and most genes known to be confined to the salmonellae are not essential for virulence. Alternative evolutionary scenarios may explain the origins of pathogenicity in Salmonella.