Initiator protein dimerization plays a key role in replication control of Vibrio cholerae chromosome 2

RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation. Mutations in rctB that reduce dimer binding increase monomer binding to 12-mers but decrease monomer binding to 39-mers. The mechanism of this paradoxical binding behavior has been unclear. Using deletion and alanine substitution mutants of RctB, we have now localized to a 71 amino acid region residues important for binding to the two kinds of DNA sites and for RctB dimerization. We find that the dimerization domain overlaps with both the DNA binding domains, explaining how changes in the dimerization domain can alter both kinds of DNA binding. Moreover, dimerization-defective mutants could be initiation-defective without apparent DNA binding defect. These results suggest that dimerization might be important for initiation beyond its role in controlling DNA binding. The finding that determinants of crucial initiator functions reside in a small region makes the region an attractive target for anti-V. cholerae drugs.     

Hepcidin is regulated during blood-stage malaria and plays a protective role in malaria infection

Hepcidin is one of the regulators of iron metabolism. The expression of hepcidin is induced in spleens and livers of mice infected with pathogenic bacteria. Recent studies have indicated that serum hepcidin level is also increased in human subjects infected with Plasmodium falciparum. The mechanism of the regulation of hepcidin expression and its role in the infection of malaria remains unknown. In this study, we determined the expression of hepcidin in livers of mice infected with Plasmodium berghei. The expression of hepcidin in the liver was upregulated and downregulated during the early and late stages of malaria infection, respectively. Inflammation and erythropoietin, rather than the iron-sensing pathway, are involved in the regulation of hepcidin expression in livers of infected mice. Meanwhile, we investigated the effect of hepcidin on the survival of mice infected with P. berghei. Treatment of malaria-infected mice with anti-hepcidin neutralizing Abs promoted the rates of parasitemia and mortality. In contrast, lentiviral vector-mediated overexpression of hepcidin improved the outcome of P. berghei infection in mice. Our data demonstrate an important role of hepcidin in modulating the course and outcome of blood-stage malaria.     

Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae

Despite over 100 years of study, the intestinal pathogen Vibrio cholerae still causes epidemic disease in areas of the world where there is poor sanitation. While cholera toxin and the toxin-coregulated pilus (TCP) are known to be essential for full virulence, the role that other factors play has remained ill-defined. Herein, we describe a large-scale signature-tagged mutagenesis (STM) screen utilizing 100 pools of 96 mutants each to identify factors involved in colonization of the infant mouse small intestine. A total of 164 mutants representing transposition events into 95 different open reading frames were shown to be recovered at greatly reduced numbers from the infant mouse model. Analysis of the sites of insertion revealed multiple independent mutations within the rfb gene cluster, needed for synthesis of lipopolysaccharide (LPS), and the tcp gene cluster, needed for synthesis of the TCP. More importantly, in addition to these previously known colonization factors, we identified many genes whose activity in colonization was not previously appreciated. These can be divided into a number of functional groups, which include production of factors involved in metabolic activities, regulation of cellular processes, transport, adaptation to stress and unknown functions. In addition, we describe the reiterative use of STM, whereby colonization-defective mutants were assembled into virulence-attenuated pools (VAPs), which were used to begin to reveal roles that the identified virulence factors play in the infection process. Nine new factors were shown to be crucial for the V. cholerae acid tolerance response, which has previously been hypothesized to be important for epidemic spread of cholera. Competition assays of these nine acid tolerance response (ATR)-defective mutants revealed that mutations in gshB, hepA and recO result in a 1000-fold reduction in colonization.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

The tcp gene cluster of Vibrio cholerae

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

The rice OsDIL gene plays a role in drought tolerance at vegetative and reproductive stages

Drought is one of the critical factors limiting reproductive yields of rice and other crops globally. However, little is known about the molecular mechanism underlying reproductive development under drought stress in rice. To explore the potential gene function for improving rice reproductive development under drought, a drought induced gene, Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein, was identified from our microarray data and selected for further study. OsDIL was primarily expressed in the anther and mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone abscisic acid (ABA). Compared with wild type, the OsDIL-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage. The expression levels of the drought-responsive genes RD22, SODA1, bZIP46 and POD, as well as the ABA synthetic gene ZEP1 were up-regulated in the OsDIL-overexpression lines but the ABA degradation gene ABAOX3 was down-regulated. Moreover, overexpression of OsDIL lessened the down-regulation by drought of anther developmental genes (OsC4, CYP704B2 and OsCP1), providing a mechanism supporting pollen fertility under drought. Overexpression of OsDIL significantly enhanced drought resistance in transgenic rice during reproductive development, while showing no phenotypic changes or yield penalty under normal conditions. Therefore, OsDIL is an excellent candidate gene for genetic improvement of crop yield in adaption to unfavorable environments.     

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L.?monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (?thiT). Cells of both wild-type and ?thiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.     

The transcriptional activator HIyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NolR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (piasmids R773, Escherichia coli; p1258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD_50.. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.     

Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection

The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter. TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth. The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro. These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions.     

Identification of a new RTX-like gene cluster in Vibrio cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism.     

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate‐dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.     

The thymus plays a role in oral tolerance in experimental autoimmune encephalomyelitis

The oral administration of myelin proteins has been used for the successful prevention and treatment of experimental autoimmune encephalomyelitis (EAE). We questioned whether the thymus was involved in oral tolerance. In this study, euthymic myelin basic protein (MBP) TCR transgenic mice are protected from EAE when fed MBP but are not protected when thymectomized. Similarly, in a cell transfer system, T cell responses to OVA measured in vivo were suppressed significantly only in the OVA-fed euthymic mice but not in the thymectomized mice. We observed that the absence of the thymus dramatically enhanced the Th1 response. We explored three alternatives to determine the role of the thymus in oral tolerance: 1) as a site for the induction of regulatory T cells; 2) a site for deletion of autoreactive T cells; or 3) a site for the dissemination of naive T cells. We found that Foxp3(+)CD4(+)CD25(+) T cells are increased in the periphery but not in the thymus after Ag feeding. These CD4(+)CD25(+) T cells also express glucocorticoid-induced TNFR and intracellular CTLA4 and suppress Ag-specific proliferation of CD4(+)CD25(-) cells in vitro. The thymus also plays a role in deletion of autoreactive T cells in the periphery following orally administered MBP. However, thymectomy does not result in homeostatic proliferation and the generation of memory cells in this system. Overall, the oral administration of MBP has a profound effect on systemic immune responses, mediated largely by the generation of regulatory T cells that act to prevent or suppress EAE.     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.