The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri

The Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations in vps or vacJ in Shigella flexneri resulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and the vpsC mutant showed minor differences in its phospholipid profile compared to the wild type. vpsC mutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase gene pldA on a multicopy plasmid in a vpsC or vacJ mutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, the pldA plasmid did not restore the mutant''s ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant''s phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system in S. flexneri in both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.     

Virulence of Shigella flexneri Hybrids Expressing Escherichia coli Somatic Antigens

The genes controlling either Escherichia coli somatic antigen 8 or 25 were conjugally transferred to virulent Shigella flexneri 2a recipients to determine whether the aquisition of these antigens would affect the virulence of the resulting hybrid. A high proportion of such hybrids were found to be rough and hence were avirulent. Some smooth S. flexneri hybrids which replaced their native group antigens with E. coli factor 25 were still virulent in the animal models employed. All S. flexneri O-8 hybrids were uniformly avirulent. Our finding, that S. flexneri hybrids with the chemically divergent E. coli O-8 repeat unit are avirulent whereas some hybrids with the chemically related O-25 repeat unit retain virulence, suggests that the chemical composition and structure of the O side chain of somatic antigens may represent one determining factor for bacterial penetration of mucosal epithelial cells, the primary step in the pathogenesis of bacillary dysentery.     

Role and regulation of iron-sulfur cluster biosynthesis genes in Shigella flexneri virulence

Shigella flexneri, a causative agent of bacterial dysentery, possesses two predicted iron-sulfur cluster biosynthesis systems called Suf and Isc. S. flexneri strains containing deletion mutations in the entire suf operon (UR011) or the iscSUA genes (UR022) were constructed. Both mutants were defective in surviving exposure to oxidative stress. The suf mutant showed growth that was comparable to that of the parental strain in both iron-replete and iron-limiting media; however, the isc mutant showed reduced growth, relative to the parental strain, in both media. Although the suf mutant formed wild-type plaques on Henle cell monolayers, the isc mutant was unable to form plaques on Henle cell monolayers because the strain was noninvasive. Expression from both the suf and isc promoters increased in iron-limiting media and in the presence of hydrogen peroxide. Iron repression of the suf promoter was mediated by Fur, and increased suf expression in iron-limiting media was enhanced by the presence of IscR. Iron repression of the isc promoter was mediated by IscR. Hydrogen peroxide-dependent induction of suf expression, but not isc expression, was mediated by OxyR. Furthermore, IscR was a positive regulator of suf expression in the presence of hydrogen peroxide and a negative regulator of isc expression in the absence of hydrogen peroxide. Expression from the S. flexneri suf and isc promoters increased when Shigella was within Henle cells, and our data suggest that the intracellular signal mediating this increased expression is reduced iron levels.     

Correlation between pathogenicity of Shigella and intraperitoneal survival in mice

Phagocytosis of virulent and avirulent strains of Shigella and Escherichia coli in the mouse peritoneum was studied. A direct correlation between bacterial virulence and resistance to phagocytosis by peritoneal phagocytes was demonstrated. Virulent strains were less readily cleared and were able to multiply to a limited extent within the peritoneal cavity. An epimerase-deficient, rough mutant of S. flexneri 2a was highly susceptible to phagocytosis. Restoration of the cell wall structure in these mutants resulted in a significant increase in their resistance to phagocytosis. Susceptibility to phagocytosis in smooth S. flexneri was age-dependent. Cells from 16-hr cultures were more resistant to removal from the peritoneum than were cells from 48- and 72-hr cultures.     

Growth of phages lambda and phiX174 under Plban protein control in the absence of host dnaB function

The absence of dnaB product in dnaB::Tn10 insertion mutants was exploited to examine the ability of coliphages λ and φX174 to use the dnaB analog protein (ban) of phage P1. A dnaB::Tn10 (P1bac) lysogen grew at 37° but not at 25° and did not support growth of λ at any temperature. Progeny phage were produced if λ infection was at high multiplicities and this production was shown to be dependent on recombination. In contrast to λ, φX174 utilized ban efficiently at all temperatures including 25°. If the level of ban was increased by using P1bac crr lysogens, the host survived at 25° and λ also grew though the latent period was extended and the rate of phage production was greatly reduced. It was also shown that mutant dnaB252, which is defective for initiation of chromosome replication, severely restricted φX174 growth at 42° and was somewhat restrictive even at 30°. Kinetic experiments demonstrated that the block to φX174 growth in this mutant was complete at 5 min after infection but by 20 min postinfection the dnaB function was no longer required.     

The NleE/OspZ family of effector proteins is required for polymorphonuclear transepithelial migration, a characteristic shared by enteropathogenic Escherichia coli and Shigella flexneri infections

Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri are human host-specific pathogens that infect intestinal epithelial cells. However, each bacterial species employs a different infection strategy within this environmental niche. EPEC attaches to the apical surface of small intestine enterocytes, causing microvillus effacement and rearrangement of the host cell cytoskeleton beneath adherent bacteria. In contrast, S. flexneri invades the large intestine epithelium at the basolateral membrane, replicates, and spreads cell to cell. Both EPEC and S. flexneri rely on type three secretion systems (T3SS) to secrete effectors into host cells, and both pathogens recruit polymorphonuclear leukocytes (PMNs) from the submucosa to the lumen of the intestine. In this report, we compared the virulence functions of the EPEC T3SS effector NleE and the homologous Shigella protein Orf212. We discovered that Orf212 was secreted by the S. flexneri T3SS and renamed this protein OspZ. Infection of polarized T84 intestinal epithelial cells with an ospZ deletion mutant of S. flexneri resulted in reduced PMN transepithelial migration compared to infection by the wild type. An nleE deletion mutant of EPEC showed a similar reduction of PMN migration. The ability to induce PMN migration was restored in both mutants when either ospZ or nleE was expressed from a plasmid. An infection of T84 cells with the ΔospZ mutant resulted in reduced extracellular signal-related kinase phosphorylation and NF-κB activation compared to infection with the wild type. Therefore, we conclude that OspZ and NleE have similar roles in the upstream induction of host signaling pathways required for PMN transepithelial migration in Shigella and EPEC infections.     

Characterization and Protective Property of Brucella abortus cydC and looP Mutants

Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.     

Salmonella Flagellin Induces Tumor Necrosis Factor Alpha in a Human Promonocytic Cell Line

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-α) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoA transposon mutagenesis was used to identify the TNF-α-inducing factor. A mutant Salmonella strain which lacks the ability to induce TNF-α was isolated from a TnphoA library. Genetic analysis of this mutant demonstrated that the hns gene has been interrupted by transposon insertion. The hns gene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 is flhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutant Salmonella strains revealed a correlation between the ability to induce TNF-α and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutant Salmonella strains to upregulate TNF-α, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition, Salmonella FliC can confer the TNF-α-inducing phenotype on Escherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-α.     

Identification of virulence genes in the crucifer anthracnose fungus Colletotrichum higginsianum by insertional mutagenesis

To investigate the molecular and genetic mechanisms underlying virulence of Colletotrichum higginsianum on Arabidopsis thaliana, a T-DNA insertion mutant library of C. higginsianum, the causal agent of crucifer anthracnose, was established using Agrobacterium tumefaciens-mediated transformation. Among 875 transformants tested for virulence on Arabidopsis, six mutants with altered virulence, including an appressorial melanin-deficient mutant T734, two mutants defective in penetration, T45 and B30, and three mutants, T679, T732 and T801, that cause hypersensitive reactions on host Arabidopsis, were obtained. Southern blot analysis indicated that the mutants T732 and T734 harbored single-site T-DNA integrations, while B30 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by inverse polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertions in mutant T734 targeted the coding region of a gene with unknown function, and in mutant T732 targeted a gene encoding a copper amine oxidase. The two T-DNA insertion sites in mutant B30 were found in the coding region of a gene encoding an exosome component and in the upstream region of a DUF221-domain gene. None of these genes have previously been implicated in virulence of the phytopathogenic fungi. Among these avirulent mutants, T734 showed altered color in colony growth and produced melanin-deficient, albino appressoria. The T-DNA insert in T734 was detected in the coding region of a gene named C. higginsianum melanin-deficiency gene (Ch-MEL1), which is highly similar to a gene encoding a hypothetical protein in Colletotrichum gloeosporioides (GenBank ELA33048). To validate whether the Ch-MEL1 gene was associated with virulence of the mutant T734, a targeted gene disruption and complementation approach was used. The appressoria of ▵Ch-mel1 null mutants were defective in melanization and failed to penetrate the host epidermal cells. When inoculated onto the wounded leaf tissues, the ▵Ch-mel1 mutants grew on host tissues but failed to cause lesions beyond the wound site. In contrast, both the complement C▵Ch-mel1-2 and the wild type produced melanized appressoria and caused necrosis on leaves of Arabidopsis. Ch-MEL1 is required for both appressorial melanin production in C. higginsianum and post-invasive growth in host tissues. Together with identification of other avirulent mutants and their associated genes, this study provides novel insights into molecular mechanisms underlying virulence of the hemibiotroph, C. higginsianum.     

Inactivation of DsbA, but Not DsbC and DsbD, Affects the Intracellular Survival and Virulence of Shigella flexneri

In this study, three mutants, dsbA::kan, dsbC-kan, and dsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, the dsbA mutant showed reduced virulence while the dsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the dsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for dsbA mutant appears to be more complicated than was previously suggested.     

A Chromosomal Locus Which Controls the Ability of Shigella flexneri to Evoke Keratoconjunctivitis

The primary step in the pathogenesis of bacillary dysentery is the penetration of intestinal epithelial cells by shigellae. Lacking this capacity, Shigella flexneri becomes avirulent. By means of intergeneric conjugation between various Escherichia coli K-12 Hfr strains and S. flexneri 2a virulent recipients and by reciprocal transduction analysis with phage P1 vir, we established a locus on the genome of S. flexneri 2a which is necessary for the ability of this strain to penetrate epithelial cells as measured by the Sereney test for keratoconjunctivitis. This locus, termed kcpA (in reference to its involvement in provoking keratoconjunctivitis), has been positioned between the lac and gal chromosomal markers and is contransducible with the purE allele.     

Roles of RseB, σE,and DegP in Virulence and Phase Variation of Colony Morphotype of Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing serious and often fatal wound infections and primary septicemia. We used alkaline phosphatase insertion mutagenesis to identify genes necessary for the virulence of this pathogen. One mutant had an in-frame fusion of ′phoA to the gene encoding RseB, a periplasmic negative regulator of the alternative sigma factor σ^E. σ^E controls an extensive regulon involved in responding to cell envelope stresses. Colonies of the rseB mutant were less opaque than wild-type colonies and underwent phase variation between translucent and opaque morphologies. rseB mutants were attenuated for virulence in subcutaneously inoculated iron-dextran-treated mice. To obtain insight into the role of rseB and the extracytoplasmic stress response in V. vulnificus, mutants with defined mutations in rseB and two important members of the extracytoplasmic stress regulon, rpoE and degP, were constructed for analysis of virulence, colony morphology, and stress-associated phenotypes. Deletion of rseB caused reversible phase variation in the colony morphotype that was associated with extracellular polysaccharides. Translucent and transparent morphotype strains were attenuated for virulence. rpoE and degP deletion mutants were sensitive to membrane-perturbing agents and heat but were not significantly attenuated for V. vulnificus virulence in mice. These results reveal complex relationships between regulation of the extracytoplasmic stress response, exopolysaccharides, and the virulence of V. vulnificus.Vibrio vulnificus is a gram-negative estuarine bacterium responsible for severe opportunistic infections (for a review, see reference 17). Ingestion of raw contaminated seafood can lead to septicemia in susceptible patients, while contact with contaminated seawater or seafood can cause wound infection, which may progress to necrotizing fasciitis and sepsis. Mortality rates for sepsis and wound infection can be as high as 75% and 50%, respectively. Predisposing conditions for septicemia include liver disease, cirrhosis, hemochromatosis, diabetes, and immune compromise, while wound infection can occur in otherwise healthy persons.Several virulence factors have been identified for V. vulnificus, most notably the antiphagocytic capsule (55, 65), RtxA toxin (26, 28, 32), and iron acquisition systems (31, 64). For a review, see reference 17. However, much is yet to be discovered, particularly the mechanisms of extreme tissue damage and rapid growth in host tissues (17). To examine these traits, we focused on the factors that are localized to the bacterial cell surface or are secreted into the extracellular space, considering that most virulence factors are exported to interact with the host. Alkaline phosphatase (phoA) mutagenesis is a useful tool for identifying genes encoding exported products (33), based on the principle that alkaline phosphatase is active only when it is exported beyond the bacterial cytoplasm. Randomly generated phoA gene fusions, most often generated via TnphoA (33), must be in genes encoding exported proteins to have enzyme activity detected by plating on the chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate (BCIP). In our studies, TnphoA did not work effectively in V. vulnificus for unknown reasons. We therefore created a mini-Tn5 transposon-based ′phoA delivery system, miniTn5phoA (8), that works well in V. vulnificus.Using this method, we identified a phoA mutant that had an in-frame fusion of ′phoA to the gene encoding RseB, a periplasmic negative regulator of sigma E (σ^E) activity. The rseB mutant exhibited several interesting phenotypes, including phase variation between translucent and opaque colony morphologies and attenuated virulence. σ^E is an alternative RNA polymerase sigma factor that controls an extensive regulon involved in responding to cell envelope stresses (48). This response, termed the extracytoplasmic stress response (ESR), is essential for maintaining the envelope integrity of gram-negative bacteria under certain stress conditions (for a review, see reference 48). Because rseB is involved in the ESR, we determined the role of the σ^E-mediated ESR in V. vulnificus. We also investigated the possible reasons for the translucent morphology of RseB variants by comparing these variants with an acapsular translucent mutant of V. vulnificus. This study uncovered a possible role for RseB in phase variation of extracellular polysaccharide (EPS) expression and is the first study to investigate the role of the ESR in the virulence of V. vulnificus.     

Isolation and characterization of chromosomal mTn10insertion mutations affecting K88 fimbriae production inEscherichia coli

mTn10transposon mutagenesis ofEscherichia coliproducing K88 fimbriae was carried out in order to identify host factors involved in the regulation of thefae(K88) operon and the production of K88 fimbriae. Five independent chromosomal insertion mutants were obtained which showed an increased expression of K88 fimbriae. Inverse PCR and nucleotide sequencing were carried out to characterize the mutations. One insertion affected thelppgene, encoding the major outer membrane lipoprotein. Another mutation was found to be located in thelrpgene, encoding the «global» regulatory protein Lrp (leucine responsive regulatory protein). A third mutant was found to affect the expression ofrfaF, encoding heptosyltransferase II, which resulted in a partially wild-type and partially R_e-R_d1type of LPS. A fourth mutation affectedsseB, a gene involved in serine-sensitivity ofE. colicells. Another mutant contained an insertion in an unknown region of theE. coligenome. The mutants were further characterized with respect to K88 as well as K99 fimbriae production.     

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.     

OmpC is involved in invasion of epithelial cells by Shigella flexneri.

Osmoregulation of the Shigella flexneri ompC gene and the role of OmpC in Shigella virulence have been investigated. OmpC was highly expressed when bacteria were grown in medium of either low or high osmolarity. This constitutive expression is in contrast with the regulation observed in Escherichia coli, in which the expression of OmpC is repressed at low osmolarity and induced at high osmolarity. In addition, the Shigella ompC gene was barely expressed by a delta ompB (delta ompR and delta envZ) mutant. We described in a previous report that such a mutant was severely impaired in virulence both in vitro and in vivo. Starting from this observation, and in order to assess which gene(s) regulated by ompR and envZ are involved in virulence, we constructed an S. flexneri delta ompC mutant. Three S. flexneri mutants, ompF'-lacZ, delta ompC, and delta ompB, were compared for virulence. The ompF'lacZ mutant behaved like the S. flexneri serotype 5 wild-type strain M90T in all in vitro and in vivo virulence tests. On the contrary, the delta ompB and delta ompC strains were considerably impaired in their virulence phenotypes. The ability of these two mutants to spread from cell to cell and to kill epithelial cells was severely affected. Consequently delta ompC, as previously described for delta ompB, was unable to elicit a positive Sereny test. The delta ompB mutant was restored to virulence by introducing a recombinant multicopy plasmid carrying the cloned E. coli ompC gene, indicating that a functional OmpC protein was necessary and sufficient to restore virulence to this mutant of S. flexneri.     

Bdellovibrio bacteriovorus Parasitism in Shigella Species

Bdellovibrio bacteriovorus was capable of parasitizing Shigella boydii, S. flexneri, and S. sonnei. The bdellovibrio was able to produce plaques on lawns consisting of heat-killed or ultraviolet-irradiated S. boydii.     

Isolation of α-toxin, θ-toxin and κ-toxin mutants ofClostridium perfringensby Tn916mutagenesis

Clostridium perfringensis the causative agent of clostridial myonecrosis or gas gangrene and mediates infection and disease by producing numerous extracellular toxins, including α-toxin, θ-toxin and κ-toxin Tn916-mutagenesis was used to isolate mutants defective in their ability to produce either α-toxin or θ-toxin. Nine independently derived mutants were isolated In four of these mutants Tn916had inserted at sites located 193 bp or 198 bp upstream of the θ-toxin structural gene,pfoAFour mutants contained large deletions, three in regions which encompassed the θ-toxin structural and regulatory genespfoAandpfoR, respectively, and the κ-toxin structural gene,colA, and one in a region encompassing the α-toxin structural gene,plcThese mutants should prove to be invaluable for further genetic studies aimed at determining the role of these toxins in virulence