Extensive cell envelope modulation is associated with virulence in Brucella abortus

Brucella virulence is linked to components of the cell envelope and tightly connected to the function of the BvrR/BvrS sensory-regulatory system. To quantify the impact of BvrR/BvrS on cell envelope proteins, we performed a label-free mass spectrometry-based proteomic analysis of spontaneously released outer membrane fragments from four strains of Brucella abortus (wild type virulent, avirulent bvrR- and bvrS- mutants as well as reconstituted virulent bvrR+ (bvrR-/pbvrR+)). We identified 167 differentially expressed proteins, of which 25 were assigned to the outer membrane. Approximately half of the outer membrane proteins decreased in abundance, whereas half increased. Notably, expression of five Omp3 family proteins decreased whereas five lipoproteins increased in the mutant strains. In the periplasmic space, by contrast, approximately 80% of the 60 differentially expressed proteins were increased in at least one avirulent mutant. Periplasmic proteins are primarily involved in substrate uptake and transport, and a uniform increase in this class may indicate a nutritional stress response, possibly a consequence of defective outer membrane function. Virtually all proteins reverted to wild type levels in the reconstituted virulent bvrR+ strain. We propose that the wide changes in cell envelope protein expression relate to the markedly avirulent phenotype of bvrR- and bvrS- mutants and that Brucella virulence depends on regulatory networks involving cell envelope and metabolism rather than on discrete virulence factors. This model may be relevant to other alpha-Proteobacteria harboring BvrR/BvrS orthologous systems known to be essential for parasitism or endosymbiosis.     

Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development

Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.     

The characteristics of antibiotic resistance and phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila

Although many typical outer-membrane proteins (OMPs) have been well characterized, the biological functions of many OMPs remain largely elusive. In this study, we successfully constructed 29 OMP knockout strains in the pathogen Aeromonas hydrophila, which account for about 50% of all predicted OMPs in this bacterial species. We then further validated the antibiotics' susceptibility characteristics against 20 antimicrobial reagents in these mutants considering several phenotypes. Our results showed that a total of 22 OMP mutants affected the susceptibility to at least one antibiotic. The deletion of some OMPs, such as ΔlamB and ΔbamA, revealed very important roles in the resistance to certain antibiotics. However, not a single OMP mutant presented a constant behaviour to all of the tested antibiotics, suggesting the existence of a complex intercellular regulation mechanism and a protein–protein interaction network underlying the OMP homeostasis in the presence of antibiotics. Meanwhile, some OMP mutants also affected biofilm formation, ECPase and haemolytic activity, and carbon resources utilization. This report demonstrates the biological functions of OMPs on a large scale and most of results have not been reported in A. hydrophila.     

Involvement and necessity of the Cpx regulon in the event of aberrant β‐barrel outer membrane protein assembly

The Cpx and σ^E regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σ^E pathway monitors the biogenesis of β‐barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β‐barrel OMP mis‐assembly, by utilizing mutants expressing either a defective β‐barrel OMP assembly machinery (Bam) or assembly defective β‐barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σ^E pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β‐barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β‐barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly‐defective β‐barrel OMP species. Together, these results showed that both the Cpx and σ^E regulons are required to reduce envelope stress caused by aberrant β‐barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.     

Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE

The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.     

The receptor channel formed by ppk25, ppk29 and ppk23 can sense the Drosophila female pheromone 7,11‐heptacosadiene

In Drosophila, pheromones play a crucial role in regulating courtship behaviors. In males, female aphrodisiac pheromones promote male‐female courtship, and male antiaphrodisiac pheromones inhibit male‐male courtship. Previous studies have reported that receptor proteins belonging to the pickpocket (ppk) family, ionotropic receptor family and gustatory receptor family are required for pheromone detection and normal courtship. However, none of them has been shown to be sufficient for sensing pheromones after ectopic expression in originally unresponsive cells. “M” cells are activated by male antiaphrodisiac pheromones but not female aphrodisiac pheromones, and the activated cells inhibit male‐male courtship. In our study, male flies with ectopic expression of ppk25, ppk29 and ppk23 in “M” cells showed decreased male‐female courtship. Using an in vivo calcium imaging approach, we found that the “M” cells expressing these three ppks were significantly activated by the female aphrodisiac pheromone 7,11‐heptacosadiene (7,11‐HD). Our results indicate that a sodium channel consisting, at minimum, of ppk25, ppk29 and ppk23, can sense 7,11‐HD, most likely as a receptor. Our findings may help us gain insights into the molecular mechanisms of pheromonal functions.     

A third envelope stress signal transduction pathway in Escherichia coli

Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The sigmaE and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The sigmaE pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.     

Deficiency of a Sinorhizobium meliloti BacA mutant in alfalfa symbiosis correlates with alteration of the cell envelope

The BacA protein is essential for the long-term survival of Sinorhizobium meliloti and Brucella abortus within acidic compartments in plant and animal cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have an increased resistance to bleomycin, it was hypothesized that BacA was a transporter of bleomycin and bleomycin-like compounds into the bacterial cell. However, our finding that the S. meliloti bacA mutant also has an increased sensitivity to detergents, a hydrophobic dye, ethanol, and acid pH supported a model in which BacA function affects the bacterial cell envelope. In addition, an S. meliloti lpsB mutant that is defective at a stage in infection of the host similar to that found for a bacA mutant is also sensitive to the same agents, and the carbohydrate content of its lipopolysaccharide (LPS) is altered. However, analysis of crude preparations of the bacA mutant LPS suggested that, unlike that for LpsB, BacA function did not affect the carbohydrate composition of the LPS. Rather, we found that at least one function of BacA is to affect the distribution of LPS fatty acids, including a very-long-chain fatty acid thought to be unique to the alpha-proteobacteria, including B. abortus.     

Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.     

A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.     

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.     

Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19

Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis.     

Modulation of tight junction barrier function by outer membrane proteins of enteropathogenic Escherichia coli: role of F-actin and junctional adhesion molecule-1

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea. In this work we investigated the effect of outer membrane proteins (OMP) of EPEC on barrier integrity and the role of actin, junctional adhesion molecule (JAM) and signaling pathways contributing to these changes. Barrier function was assessed by transepithelial electrical resistance (TER). OMP of wild type EPEC, eaeA and maltoporin mutants decreased TER levels of Caco-2 cells. The OMP of espB mutant was deficient in decreasing TER of Caco-2 cells. The proteinase K-digested wild type OMP and EAF mutant OMP did not cause any change in barrier function. Our previous studies have demonstrated that EPEC OMP induced changes in cadherin junctions of Caco-2 cells. Immunofluorescence revealed disruption in actin cytoskeleton by EPEC OMP. However, no change in expression of junctional adhesion molecule-1 was observed. NF-kappaB inhibitor slightly blocked the decrease in TER and protected against actin disruption while ERK1/2 inhibitor had no effect in blocking these changes. In conclusion, our data suggest that the OMP of EPEC alter intestinal barrier function by disrupting actin cytoskeleton and signaling pathways like NF-kappaB may have a role in regulating barrier changes.