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Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways. The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function. Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas. B. pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion. Here we review current understanding of the molecular mechanisms underlying these processes.

We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2. Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion. Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for Cdc42 and Rac1 in vitro.

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.

The Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella-containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella-induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI-2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wild-type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.

Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)-1ss, IL-2, IL-6 and interferon (IFN)-gamma were measured by quantitative RT-PCR, and production of IL-6 and IFN-gamma was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-gamma compared with non-infected cells; S. enteritidis invasion caused a slight down-regulation of IL-2 production. For IL-1ss production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1ss mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of 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.

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.

The YopJ protein of Yersinia pseudotuberculosis inhibits several eukaryotic signalling pathways that are normally activated in cells following their contact with bacteria. Salmonella encodes a protein, AvrA, that is secreted by the typeIII inv/spa secretion system which is clearly homologous to YopJ (56% identical, 87% similarity). Since AvrA and YopJs similarity also encompassed a region of YopJ that had previously been shown to be critical for its biological activity, we were interested whether AvrA and YopJ provoked similar responses in eukaryotic cells. Two different approaches were used to determine whether AvrA possesses YopJ-like activity in modulating cytokine expression or killing macrophages. An avrA strain of Salmonella dublin was constructed and its activity was compared to an isogenic wildtype counterpart in cellular response assays. In a complementary approach, AvrA was expressed in and delivered into eukaryotic cells by a yopJ strain of Yersinia pseudotuberculosis. We show here that AvrA affects neither cytokine expression or plays a role in macrophage killing when expressed by either Salmonella or Yersinia. Additionally, AvrA does not possess SopB/D-like activity in promoting fluid secretion into infected calf ileal loops. These data indicate that Salmonella and Yersinia trigger and/or modulate eukaryotic cell responses by different typeIII-secreted proteins and suggests that despite their close evolutionary relatedness, AvrA and YopJ perform different functions for Salmonella and Yersinia, respectively., (Copyright 2000 Academic Press.)

The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective., (Copyright 1999 Academic Press.)

The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in Salmonella-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of Salmonella spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the sopB gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of Salmonella dublin. Using the cya reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of sopD has an additive effect in relation to the sopB mutation.

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5, 6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456-14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.

Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella-specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA1ser (serT) on one side and copS/copR on the other. Thus, this Salmonella-specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA, pipB, pipC, pipD (pathogenicity island-encoded proteins) and orf, in addition to sopB. The effect of mutations in pipA, pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.

The induction of secretory and inflammatory responses in calves by Salmonella typhimurium and Salmonella dublin strains was compared, and the effects of mutations in the invH and stn genes were assessed. S. typhimurium induced greater secretory and inflammatory responses than S. dublin in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P. R. Watson, S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis, Infect. Immun. 63:2743-2754, 1995). These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis. The secretory and inflammatory responses induced by S. typhimurium and S. dublin in bovine ligated ileal loops were not significantly altered by mutation of stn, which suggests that stn does not have a major role in Salmonella-induced enteritis. The invH mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves. The attenuation associated with the invH mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of S. typhimurium to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered. However, lysis of macrophages following infection with S. typhimurium was significantly reduced by the invH mutation. The invH mutation prevented the normal secretion of several proteins, including SipC, by S. typhimurium, indicating that the function of the inv-spa-encoded type III protein secretion system was disrupted. Taken together, these observations implicate inv-spa-dependent effectors in mediation of Salmonella-induced enteritis in cattle. Clearly, however, other undefined serotype-specific virulence factors are also involved in Salmonella-induced enteritis.

Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.

The entry of Salmonella into cultured epithelial cells is dependent on genes located in several adjacent chromosomal loci. One of these loci encodes the recently identified secretory proteins, denoted Sips (Salmonella invasion proteins). SipB, C,D proteins are essential for the ability of the pathogen to invade epithelial cells. To examine if additional invasion-associated proteins were secreted by Salmonella dublin, the genes encoding already characterized secretory proteins were inactivated to facilitate this analysis. The proteins produced and secreted by a double fIIM/polar sipB mutant of S. dublin were analysed; this revealed a set of novel secreted proteins. These proteins, which we denoted Sops (Salmonella outer proteins), formed large filamentous aggregates in the medium of bacterial culture growing at 37 degrees C. These aggregates contained five predominant proteins. Here we report the identification and characterization of one of these proteins, SopE, which is a novel invasion-associated secretory protein of S. dublin. A specific sopE mutant of S. dublin was found to be defective for invasion into epithelial cells. Upon interaction of Salmonella with HeLa cells, SopE was found to be translocated into the cytoplasm of the target cell by extracellular bacteria. The translocation of SopE was shown to be dependent on the Sip proteins because a polar sipB mutant did not translocate SopE across the HeLa cell membrane.

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high-copy-number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild-type strain with respect to YopB-dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB-dependent translocation mechanism from surface-located bacteria and subsequently targeted to the inner surface of the target-cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.

The Ser/Thr protein kinase YpkA, encoded by the virulence plasmid pIB1, is an indispensable virulence determinant of Yersinia pseudotuberculosis [E. E. Galyov, S. Håkansson, A. Forsberg, and H. Wolf-Watz, Nature (London) 361:730-732, 1993]. In this study, the organization of the ypkA-containing operon and the in vitro regulation of this transcriptional unit were characterized. The operon contains two structural genes, ypkA and yopJ, and is regulated by temperature and the extracellular concentration of Ca2+, as are the yop genes. The two proteins were secreted without posttranslational processing, showing that YpkA and YopJ belong to the Yop family. Mutational analysis revealed that, in contrast to all other Yop proteins so far studied, the YopJ protein was dispensable for virulence of Y. pseudotuberculosis.

Phosphorylation of proteins catalysed by protein kinases is associated with central functions in growth and proliferation of the eukaryotic cell, and kinases are particularly important in the signal transduction pathways. Enterobacterial protein kinases are structurally and functionally different from eukaryotic protein kinases, and no prokaryotic kinase has so far been described implicating a direct role for this activity in virulence. Virulent Yersinia possess a common virulence plasmid that encodes a number of secreted proteins (Yops), of which YopH has protein-tyrosine phosphatase activity with a key function in the block of phagocytosis by the pathogen. Here we report that the virulence plasmid of Yersinia pseudotuberculosis encodes a secreted protein kinase (YpkA) with extensive homology to eukaryotic Ser/Thr protein kinases. Specific mutants of ypkA resulted in avirulent strains. Thus, YpkA is, to our knowledge, the first reported prokaryotic secreted protein kinase involved in pathogenicity, presumably by interfering with the signal transduction pathways of the target cell.

A new transcription unit of the f1 gene cluster was found. The DNA sequencing revealed one long open reading frame. Deletion and frame shift mutation analyses have demonstrated the importance of a corresponding gene product for the F1 antigen biosynthesis. A homology of the deduced amino acid sequence with that of AraC family DNA-binding regulators was shown. A potential regulatory DNA region is discussed.

The DNA sequence determination of the f1 operon between the genes encoding the F1 subunit (caf1) and chaperone-like protein (caf1M) revealed a large open reading frame that codes for a polypeptide similar to some E. coli proteins involved in the biogenesis of fimbria. The deletion and in trans complementation analyses showed that this gene is not necessary for extracellular transport of the F1 subunit but plays a role in the capsule assembly.

Over 99% of the genome of Aedes densonucleosis virus was determined. Two types of the viral DNA were found that differ only in four nucleotides (nt) in the 5' noncoding part and whose sizes are 4009 nt (more copious) and 4012 nt, respectively. Both 146 nt at the 3' end and 164 nt at the 5' end could assume a similar T-shaped structure; but unlike the adeno-associated virus, Aedes DNA has a unique primary DNA sequence at each terminus. However, the crossarms of these structures are built of the same sequences. An imperfect direct repeat of 34 nt was observed in the 5' noncoding part. The plus strand has three large open reading frames (ORF): a left ORF, a right ORF, and a mid ORF (within the left ORF). The left ORF codes for the nonstructural protein NS-1 (97.5K) featured by an NTP-binding domain, and the right ORF encodes the both capsid proteins, the smaller of which (39K) is supposed to be derived from the larger one (40.5K) by proteolytic cleavage. There is also an ORF in the minus strand. The putative polypeptide coded by this ORF is extremely hydrophobic.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the caf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively.

The plasmid-located gene caf1 encoding the capsular antigen fraction 1 (F1) of Yersinia pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced Mr of 17.6 kDa. The signal peptide sequence was highly homologous to the E. coli consensus signal sequence. The F1 was assumed to have beta-sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells.