Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.     

Role of HtrA in surface protein expression and biofilm formation by Streptococcus mutans

The HtrA surface protease in gram-positive bacteria is involved in the processing and maturation of extracellular proteins and degradation of abnormal or misfolded proteins. Inactivation of htrA has been shown to affect the tolerance to thermal and environmental stress and to reduce virulence. We found that inactivation of Streptococcus mutans htrA by gene-replacement also resulted in a reduced ability to withstand exposure to low and high temperatures, low pH, and oxidative and DNA damaging agents. The htrA mutation affected surface expression of several extracellular proteins including glucan-binding protein B (GbpB), glucosyltransferases, and fructosyltransferase. In addition, htrA mutation also altered the surface expression of enolase and glyceraldehyde-3-phosphate dehydrogenease, two glycolytic enzymes that are known to be present on the streptococcal cell surface. As expected, microscopic analysis of in vitro grown biofilm structure revealed that the htrA deficient biofilms adopted a much more granular patchy appearance, rather than the relatively smooth confluent layer normally seen in the wild type. These results suggest that HtrA plays an important role in the biogenesis of extracellular proteins including surface associated glycolytic enzymes and in biofilm formation of S. mutans.     

Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant.

In order to evaluate the biological function of the Brucella abortus high-temperature-requirement A (HtrA) stress response protein homolog, the majority of the htrA gene was deleted from the chromosome of B. abortus 2308 via gene replacement. In contrast to the parental strain, the resulting htrA deletion mutant, designated PHE1, failed to grow on solid medium at 40 degrees C and demonstrated increased sensitivity to killing by H2O2 and O2- in disk sensitivity assays. BALB/c mice were infected with strains 2308 and PHE1 to assess the effect of the htrA mutation on virulence, and significantly fewer brucellae were recovered from the spleens of mice infected with PHE1 than from those of mice infected with 2308 at 1 week postinfection. Genetic complementation studies were performed to confirm the relationship between the htrA mutation and the phenotype observed for PHE1. Plasmid pRIE1 was constructed by inserting a 1.9-kb EcoRI fragment encoding the B. abortus htrA gene into the broad-host-range plasmid pBBR1MCS. Introduction of pRIE1 into PHE1 relieved the temperature- and H2O2-sensitive phenotypes of this mutant in vitro, and PHE1(pRIE1) colonized the spleens of BALB/c mice at levels equivalent to those of the parental 2308 strain at 1 week postinfection. These results support our previous proposal that the B. abortus htrA gene product functions as a stress response protein and further suggest that this protein contributes to virulence. These studies also demonstrate the utility of the broad-host-range plasmid pBBR1MCS for genetic complementation studies in Brucella spp., establishing a key reagent for more detailed genetic analysis of this important zoonotic pathogen.     

HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells

Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103mapstoR and S212mapstoA) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrA promoter to probe the phagosomal microenvironment harboring L. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.     

Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S

The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37 degrees C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria.     

Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice

A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrA mutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortus RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonella and Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.     

Role of the htrA gene in Klebsiella pneumoniae virulence

We recently described the use of mini-Tn5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50 degrees C) and oxidative stress (H(2)O(2)) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae.     

The mgtC gene of Burkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages

Burkholderia cenocepacia, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which B. cenocepacia causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that B. cenocepacia prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify B. cenocepacia genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the mgtC gene. Here, we show that mgtC is also needed for growth of B. cenocepacia in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that B. cenocepacia mgtC mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection, mgtC mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an mgtC mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.     

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.     

Cable pili and the 22-kilodalton adhesin are required for Burkholderia cenocepacia binding to and transmigration across the squamous epithelium

Burkholderia cenocepacia strains expressing both cable (Cbl) pili and the 22-kDa adhesin bind to cytokeratin 13 (CK13) strongly and invade squamous epithelium efficiently. It has not been established, however, whether the gene encoding the adhesin is located in the cbl operon or what specific contribution the adhesin and Cbl pili lend to binding and transmigration or invasion capacity of B. cenocepacia. By immunoscreening an expression library of B. cenocepacia isolate BC7, we identified a large gene (adhA) that encodes the 22-kDa adhesin. Isogenic mutants lacking expression of either Cbl pili (cblA or cblS mutants) or the adhesin (adhA mutant) were constructed to assess the individual role of Cbl pili and the adhesin in mediating B. cenocepacia binding to and transmigration across squamous epithelium. Relative to the parent strain, mutants of Cbl pili showed reduced binding (50%) to isolated CK13, while the adhesin mutant showed almost no binding (0 to 8%). Mutants lacking either cable pili or the adhesin were compromised in their ability to bind to and transmigrate across the squamous epithelium compared to the wild-type strain, although this deficiency was most pronounced in the adhA mutant. These results indicate that both Cbl pili and the 22-kDa adhesin are necessary for the optimal binding to CK13 and transmigration properties of B. cenocepacia.     

Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence

The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. A Listeria monocytogenes 10403S htrA mutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.     

Effect of inactivation of the HtrA-like serine protease DegQ on the virulence of Salmonella enterica serovar Typhimurium in mice

DegQ is a serine protease that is highly homologous to HtrA, an important virulence determinant of Salmonella enterica serovar Typhimurium. We examined if DegQ is involved in serovar Typhimurium pathogenesis. A serovar Typhimurium degQ mutant was as virulent as the wild-type strain in mice. However, a serovar Typhimurium htrA degQ mutant survived less well in murine organs, particularly in the liver, than a serovar Typhimurium htrA mutant. DegQ is not essential for serovar Typhimurium pathogenesis but may play a small role during salmonella growth at systemic sites.     

Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans

Burkholderia cenocepacia H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by cep, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode Caenorhabditis elegans, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the cep-regulated virulence factors required for nematode killing. Here we report the identification of a cep-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen Ralstonia solanacearum. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that B. cenocepacia H111 AidA is essential for slow killing of C. elegans but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by B. cenocepacia but also by many other strains of the Burkholderia cepacia complex.     

Impaired resistance to infection does not increase the virulence of Salmonella htrA live vaccines for mice.

We have described a new class of live attenuated salmonella vaccines harbouring lesions in htrA, a stress protein gene previously. The virulence and invasiveness of Salmonella htrA mutants was investigated in three models of increased susceptibility to Salmonella infection. These included BALB/c mice, either given sublethal whole body irradiation (350 R) or administered rabbit anti-TNF alpha antiserum, and (CBA/NfemaleXBALB/cmale)F1 male mice which express the xid sex-linked B cell defect of CBA/N mice and are more susceptible to salmonellae than female littermates. Salmonella typhimurium htrA mutants derived from virulent strains, C5046 (C5 htrA::TnphoA) and BRD726 (SL1344 delta htrA) were not more invasive in immunosuppressed mice than in normal controls in the three mouse models of defective immunity. The results indicate that susceptibility to S. typhimurium htrA vaccines derived from virulent parents is not enhanced by conditions of impaired resistance to infection.     

Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation

Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients. To understand the contribution of B. cenocepacia flagella to infection, a strain mutated in the major flagellin subunit, fliCII, was constructed in B. cenocepacia K56-2 and tested in a murine agar bead model of lung infection. C57/BL6 mice infected with approximately 10(8) wild-type K56-2 bacteria exhibited 40% mortality after 3 days, whereas no mortality was noted in mice infected with the fliCII mutant. Among the mice surviving the infection with either strain, there was no significant difference in the bacterial loads in the lungs and spleen, bacteremia, weight loss, or infiltration of immune effector cells at 3 days postinfection. Similar results were observed at 24 h, prior to expression of the lethality phenotype. KC, a murine interleukin-8 (IL-8) homolog, was elevated in both the bronchoalveolar lavage fluid and serum of mice infected with the wild type compared to the fliCII mutant at 24 h, suggesting that flagella stimulated host cells. To demonstrate that flagella contributed to these responses, the interaction between B. cenocepacia and Toll-like receptor 5 (TLR5) was investigated. Infection of HEK293 cells with heat-killed wild-type K56-2, but not infection with the fliCII mutant, resulted in both NF-kappaB activation and IL-8 secretion that was dependent upon expression of TLR5. Together, these results demonstrate that B. cenocepacia flagella contribute to virulence in an in vivo infection model, and that induction of host immune responses through interaction with TLR5 may contribute to its overall pathogenic potential.     

The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella typhimurium

In Escherichia coli, extracytoplasmic stress is partially controlled by the alternative sigma factor, RpoE (sigmaE). In response to environmental stress or alteration in the protein content of the cell envelope, sigmaE upregulates the expression of a number of genes, including htrA. It has been shown that htrA is required for intramacrophage survival and virulence in Salmonella typhimurium. To investigate whether sigmaE-regulated genes other than htrA are involved in salmonella virulence, we inactivated the rpoE gene of S. typhimurium SL1344 by allelic exchange and compared the phenotype of the mutant (GVB311) in vitro and in vivo with its parent and an isogenic htrA mutant (BRD915). Unlike E. coli, sigmaE is not required for the growth and survival of S. typhimurium at high temperatures. However, GVB311 did display a defect in its ability to utilize carbon sources other than glucose. GVB311 was more sensitive to hydrogen peroxide, superoxide, and antimicrobial peptides than SL1344 and BRD915. Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant was defective in its ability to survive and proliferate in both cell lines. The effect of the rpoE mutation on the intracellular behavior of S. typhimurium was greater than that of the htrA mutation. Both GVB311 and BRD915 were highly attenuated in mice. Neither strain was able to kill mice via the oral route, and the 50% lethal dose (LD50) for both strains via the intravenous (i.v.) route was very high. The i.v. LD50s for SL1344, BRD915, and GVB311 were <10, 5.5 x 10(5), and 1.24 x 10(7) CFU, respectively. Growth in murine tissues after oral and i.v. inoculation was impaired for both the htrA and rpoE mutant, with the latter mutant being more severely affected. Neither mutant was able to translocate successfully from the Peyer's patches to other organs after oral infection or to proliferate in the liver and spleen after i.v. inoculation. However, the htrA mutant efficiently colonized the livers and spleens of mice infected i.v., but the rpoE mutant did not. Previous studies have shown that salmonella htrA mutants are excellent live vaccines. In contrast, oral immunization of mice with GVB311 was unable to protect any of the mice from oral challenge with SL1344. Furthermore, i.v. immunization with a large dose ( approximately 10(6) CFU) of GVB311 protected less than half of the orally challenged mice. Thus, our results indicate that genes in the sigmaE regulon other than htrA play a critical role in the virulence and immunogenicity of S. typhimurium.     

Burkholderia cepacia complex epidemiology in persons with cystic fibrosis from Australia and New Zealand

The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.