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Genomic approaches to understanding bacterial virulence.

The genomic sequences of bacterial pathogens and of the host species they infect have greatly increased the understanding of host-pathogen interactions. Sequences of bacterial genomes have led to the identification of virulence factors through the use of bioinformatics, targeted mutant library construction, screening approaches combining transposon mutagenesis and microarray technology, and through the expression of libraries of bacterial proteins within model organisms such as yeast. Host genomic information has also yielded insights into bacterial virulence through transcriptional profiling of host responses to infection and identification of host proteins required for bacterial pathogenicity using knockdown of host gene product expression during infection. Research using genomic approaches to bacterial pathogenesis is a rapidly growing field and will expand further as additional bacterial genome sequences become available and techniques for conducting high-throughput analysis are refined.

Bacteria↗

Bacillus subtilis DegU acts as a positive regulator for comK expression.

Bacillus subtilis ComK plays a critical role in competence development. We report that B. subtilis degR, a positive regulator for exoenzyme production, is negatively regulated by overproduced ComK caused by a mecA null mutation. To identify a positive regulator for comK expression in the mecA background, mutations that allowed the degR gene to be expressed were screened in Tn10 transposon insertion mutants. As a result, we identified degU insertion mutations as those having such a property. The degU mutation reduced comK-lacZ expression in a competence medium in both the wild-type and mecA cells in sporulation and competence media. These results indicate that the degU gene product acts as a positive regulator for comK expression even under the condition where the negative regulation of comK by MecA is released.

Bacillus subtilis↗

Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio.

BACKGROUND: Transposable elements of the Tc1/mariner family are found in many species of the animal kingdom. It has been suggested that the widespread distribution of this transposon family resulted from horizontal transmission among different species. RESULTS: To test the ability of Tc1/mariner to cross species barriers, as well as to develop molecular genetic tools for studying zebrafish development, we determined the ability of the Tc3 transposon, a member of the Tc1/mariner family, to function in zebrafish. Tc3 transposons carrying sequences encoding the green fluorescent protein (GFP) were able to integrate in the fish genome by transposition. Integrated transposons expressed the GFP marker after germline transmission, and were capable of being mobilized upon introduction of transposase protein in trans. CONCLUSIONS: Our findings support models of horizontal transmission of Tc1/mariner elements between species. The work also establishes the basis for a novel method of transposon-mediated genetic transformation and for transposon-mediated genetic screens in zebrafish and other organisms.

Animals↗

Multiply antibiotic-resistant Vibrio cholerae O1 biotype El Tor strains emerge during cholera outbreaks in Zambia.

Antibiotic resistance data, made available from laboratory records during eight cholera outbreaks between 1990 and 2004 showed Vibrio cholerae serogroup O1 to have a low level of resistance (2-3%) to tetracycline during 1990-1991. Resistance increased for tetracycline (95%), chloramphenicol (78%), doxycycline (70%) and trimethoprim-sulphamethoxazole (97%) in subsequent outbreaks. A significant drop in resistance to tetracycline and chloramphenicol followed the adoption of a national policy to replace tetracycline with erythromycin for treating cholera. Sixty-nine strains from cholera outbreaks in Zambia between 1996 and 2004, were examined for antibiotic resistance and basic molecular traits. A 140 MDa conjugative, multidrug-resistant plasmid was found to encode tetracycline resistance in strains from 1996/1997 whereas strains from 2003/2004 were resistant to furazolidone, but susceptible to tetracycline, and lacked this plasmid. PCR revealed 25 of 27 strains from 1996/1997 harboured the intl1 class 1 integron but lacked SXT, a conjugative transposon element. Similar screening of 42 strains from 2003/2004 revealed all carried SXT but not the intl1 class 1 integron. All 69 strains, except two, one lacking ctxA and the other rstR and thus presumably truncated in the CTX prophage region, were positive for important epidemic markers namely rfbO1, ctxA, rstR2, and tcpA of El Tor biotype. Effective cholera management is dependent on updated reports on culture and sensitivity to inform the choice of antibiotic. Since the emergence of antibiotic resistance may significantly influence strategies for controlling cholera, continuous monitoring of epidemic strains is crucial.

Bacterial Typing Techniques↗

The fadD2 gene is required for efficient Mycobacterium avium invasion of mucosal epithelial cells.

OBJECTIVE: Mycobacterium avium is capable of invading the intestinal epithelial cells, which requires cytoskeleton rearrangement and protein phosphorylation in the host cell. However, little is known about the mechanism. METHODS: A transposon bank was screened for invasion-impaired mutants. RESULTS: Among the genes identified, inactivation of the fadD2 gene resulted in approximately 50% reduction in invasion in vitro and 100-fold reduction in invasion in vivo, compared with the wild-type (wt) strain. Invasion by wt M. avium led to the recruitment of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which was not observed with mutant lacking a functional fadD2 gene. M. avium entry resulted in the phosphorylation of N-WASp and activation of the Arp2/3 complex. Supernatant obtained from wt M. avium incubated with HEp2 epithelial cells rescued the mutant 1B2 ability to enter the cells, which suggests that activation of Cdc42 probably follows the secretion of M. avium proteins. CONCLUSION: fadD2 is a regulator of M. avium invasion, and its effect is through Cdc42.

Actin-Related Protein 2-3 Complex↗

Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness.

A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.

Adaptation, Physiological↗

Multiresistance genes of Rhizobium etli CFN42.

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.

Amino Acid Sequence↗

Identification of an extracellular matrix protein adhesin, EmaA, which mediates the adhesion of Actinobacillus actinomycetemcomitans to collagen.

Actinobacillus actinomycetemcomitans is an aetiologic agent in the development of periodontal and some systemic diseases in humans. This pathogen localizes to the underlying connective tissue of the oral cavity in individuals with periodontal disease. The adhesion of A. actinomycetemcomitans to extracellular matrix components of the connective tissue prompted this study to identify gene products mediating the interaction of A. actinomycetemcomitans to these molecules. A transposon mutagenesis system was optimized for use in A. actinomycetemcomitans and used to generate an insertional mutant library. A total of 2300 individual insertion transposon mutants were screened for changes in the adhesion to collagen and fibronectin. Mutants were identified which exhibited the following phenotypes: a decrease in collagen binding; a decrease in fibronectin binding; a decrease in binding to both proteins; and an increase in binding to both collagen and fibronectin. The identification of mutants defective in adhesion to the individual proteins indicates that distinct adhesins are expressed by this organism. Molecular analysis of these mutants implicated 11 independent loci in protein adhesion. One gene, emaA, is likely to encode a direct mediator of collagen adhesion, based on predicted protein features homologous to the collagen-binding protein YadA of Yersinia enterocolitica. EmaA was localized to the outer membrane, as expected for an adhesin. Reduction in fibronectin adhesion appeared to be influenced by abrogation of proteins involved in molybdenum-cofactor biosynthesis. Several other loci identified as reducing or increasing adhesion to both collagen and fibronectin are suggested to be involved in regulatory cascades that promote or repress expression of collagen and fibronectin adhesins. Collectively, the results support the hypothesis that A. actinomycetemcomitans host colonization involves afimbrial adhesins for extracellular matrix proteins, and that the expression of adhesion is modulated by global regulatory mechanisms.

Adhesins, Bacterial↗

The Caenorhabditis elegans SH2 domain-containing protein tyrosine phosphatase PTP-2 participates in signal transduction during oogenesis and vulval development.

Src homology-2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) have been identified as either positive or negative regulators of signaling events downstream of receptor protein tyrosine kinases (R-PTKs). We describe here our characterization of ptp-2, a Caenorhabditis elegans gene that encodes a 668-amino-acid SHP. We isolated a recessive ptp-2 loss-of-function allele, op194, that lacks the conserved protein tyrosine phosphatase catalytic domain by screening for transposon-mediated deletion mutations. Homozygous ptp-2(op194) hermaphrodites exhibit a completely penetrant zygotic semisterile/maternal effect lethal phenotype, characterized by the presence of abnormally large oocytes in the zygotic semisterile animals. These phenotypes indicate that PTP-2 activity is essential for proper oogenesis. Gain-of-function let-60 ras alleles rescued the defects associated with ptp-2(op194), suggesting that LET-60 Ras acts downstream of, or in parallel to, PTP-2 during oogenesis. Although ptp-2 function is not required for normal vulval development, ptp-2(op194) altered significantly the vulval phenotypes caused by mutations in several genes of the inductive signaling pathway. The penetrance of the multivulva phenotype caused by loss-of-function mutations in lin-15, and gain-of-function mutations in let-23 or let-60 ras, was reduced by ptp-2(op194). Moreover, ptp-2(op194) increased the penetrance of the vulvaless phenotype conferred by a weak loss-of-function sem-5 allele. Taken together, our genetic data positions PTP-2 activity downstream of LET-23 in the vulval induction signaling pathway. Although PTP-2 functions to transmit a requisite signal during oogenesis, PTP-2 function during C. elegans vulval cell differentiation appears to be directed at regulating the overall strength of the inductive signal, which may contribute to the quantitative differences in signaling required for the proper specification of the 1 degrees , 2 degrees , and 3 degrees vulval cell fates.

Alleles↗

LAF3, a novel factor required for normal phytochrome A signaling.

Phytochrome A (phyA) is the photolabile plant light receptor that mediates broad spectrum very low-fluence responses and high irradiance responses to continuous far-red light (FRc). An Arabidopsis mutant laf3-1 (long after far-red 3) was recovered from a screen for transposon-tagged mutants that exhibit reduced inhibition of hypocotyl elongation in FRc. The laf phenotype correlated well with a strongly attenuated disappearance of XTR7 transcript in FRc. The effects of laf3-1 on phyA-controlled CAB, CHS, and PET H expression were more subtle, and the mutation had no clear effects on PET E and ASN1 transcript levels in FRc. The use of two alternative transcription initiation sites in the LAF3 gene generates two isoforms that differ only at their N termini. Transcripts encoding both isoforms were induced during germination and were present at slightly higher levels in de-etiolated seedlings than in those grown in darkness. No significant differential regulation of the two isoforms was observed upon exposure to either FRc or continuous red light. Transcripts encoding the shorter isoform (LAF3ISF2) always appear to be more abundant than those encoding the longer isoform (LAF3ISF1). However, both isoforms were capable of full complementation of the laf3-1 hypocotyl phenotype in FRc. When fused to a yellow fluorescent protein, both isoforms localize to the perinuclear region, suggesting that LAF3 encodes a product that might regulate nucleo-cytoplasmic trafficking of an intermediate(s) involved in phyA signal transduction.

Arabidopsis↗

Cloning and characterization of the genes encoding the hemolysin of Haemophilus ducreyi.

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi. In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35,000-1, was further characterized. Sequences flanking the Tn916 element in strain 35,000-1 were employed to identify clones from a lambda DASHII library of H. ducreyi strain 35,000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35,000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB, were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens.

Amino Acid Sequence↗

Investigating the role of secA2 in secretion and glycosylation of a fimbrial adhesin in Streptococcus parasanguis FW213.

Adhesion of Streptococcus parasanguis FW213, a primary colonizer, to the tooth surface is mediated mainly by peritrichous long fimbriae. The fimbrial structural unit, Fap1, is indispensable for fimbriae biogenesis, adhesion to an in vitro tooth model and biofilm formation. Mature Fap1 is a glycoprotein with an apparent molecular mass of 200 kDa. Glycosylated Fap1 is not present in some mutants screened from a transposon mutant library. Localization of the transposition sites revealed a gene determined to be secA2, which is distinct from the canonical secA gene. In FW213, glycosylated Fap1 was present in all the subcellular fractions including the cytoplasm. In VT1574, a non-polar mutant of secA2 generated by in frame deletion, Fap1 was not secreted. Glycosylated Fap1 was present in the membrane and cytoplasm of the mutant, although in greatly reduced amounts. Fap1 secretion and abundance were restored when VT1574 was complemented by a plasmid-borne secA2. The secretion defect of the secA2 mutation appears to be limited to a small group of proteins such as Fap1 and FimA. These data suggested that Fap1 secretion rather than glycosylation was the major effect of the deletion of secA2; however, this deletion also had an impact on Fap1 abundance. Two more secA2 mutants with different regions deleted were tested for their ability to secrete Fap1. One mutant was completely unable to secrete Fap1 while the other was able to secrete, but in a decreased amount. These data suggest that the region deleted in the latter mutant (nucleotides 2032-2337) is dispensable for Fap1 secretion.

Adenosine Triphosphatases↗

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase.

Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

Electron Transport Complex IV↗

Isolation and characterization of a genetically tractable photoautotrophic Fe(II)-oxidizing bacterium, Rhodopseudomonas palustris strain TIE-1.

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H2, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (alpha-FeOOH) forms, and above pH 7.2 magnetite (Fe3O4) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of approximately 10(-5). To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis.

Carbon↗

Genetic and biochemical evidence for the involvement of a molybdenum-dependent enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106.

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.

Coenzymes↗

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

Bordetella bronchiseptica↗

Identification and characterization of a phase-variable nonfimbrial Salmonella typhimurium gene that alters O-antigen production.

Salmonella typhimurium 798, which was isolated from a pig, is known to phase vary from a nonadhesive to an adhesive phenotype. Cells of the adhesive phenotype adhere to porcine enterocytes, are more readily phagocytized by porcine neutrophils and macrophages, and once phagocytized can survive intracellularly, while cells of the nonadhesive phenotype die rapidly. The effect of phenotypic switching also can be visualized by changes in colony morphologies and the presence of between 10 and 15 proteins in the envelopes of cells in the adhesive phenotype. Mutants previously constructed with cells in the adhesive phenotype and the transposon TnphoA were screened to identify mutants lacking one or more of the unique proteins. One mutation was cloned and sequenced, and the mutation was shown to be in rfaL (O-antigen ligase). Expression of O antigen was shown to be phase variable. The adhesive strain expressed an O antigen that was at least eightfold longer than that for the nonadhesive strain and by virtue of O-antigen production was resistant to porcine complement. The mutant survived intracellularly in phagocytic cells as well as its wild-type parent.

Animals↗

Role of flagella in host cell invasion by Burkholderia cepacia.

Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of B. cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of B. cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of B. cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B. cepacia. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B. cepacia, contributing to establishment of infection and systemic spread of the organism.

Bacterial Adhesion↗