Effects of nutrient restriction during early or mid-gestation in bovine on placental development and miRNA expression in the cotyledon.

The objective of this study was to determine effects of maternal nutrient restriction (NR) during early or mid-gestation on uterine composition and miRNA expression in cotyledons. Primiparous Angus-cross cows (n = 38) were synchronized and inseminated using male sexed semen, blocked by body condition score and body weight (BW), and assigned to treatments. Animals were fed either: control (CON; gain 1 kg/week) or NR (55% maintenance energy and crude protein requirements) based on BW. An initial set of animals were fed either NR (n = 8) or CON (n = 8) from day 30-110 of gestation. A second set of animals were fed CON (n = 8) d 30-190 (CON/CON); NR (n = 7) day 30-110 followed by CON day 110-190 (NR/CON); or CON (n = 7) day 30-110 followed by NR day 110-190 (CON/NR). Cows were harvested on day 110 or 190 of gestation to collect placental tissues. RNA was isolated from cotyledon samples (3 animals/group) prior to microarray analysis using known Bos taurus microRNA sequences. Relative microRNA abundance was analyzed via ANOVA. Maternal NR increased (P < 0.05) cotyledon weight and total placentome surface area irrespective of gestational day. At day 110 of gestation, 51 microRNAs were reduced while 91 microRNAs observed greater abundance (P < 0.05) in NR verses CON cotyledons. At day 190 of gestation, 40 microRNAs were reduced and 26 microRNAs were increased (P < 0.05) in both NR/CON and CON/NR verses CON cotyledons. Top KEGG pathway analysis included: axon guidance, endocytosis, neuroactive ligand receptor interaction, and MAPK signaling pathway. Early-gestation maternal NR altered microRNA abundance to a greater extent than mid-gestation NR.

The effects of early- or mid-gestation nutrient restriction on bovine fetal pancreatic development.

Research on the effects of nutrient restriction in beef cows on fetal pancreatic development is limited. To address this, multiparous Angus-cross cows (n = 22) were fed either control (CON; to gain 1 kg/wk) or nutrient-restricted (NR; 0.55% NEm) diets based on NRC requirements. On d 30 of gestation, cows were blocked by body condition and randomly assigned to one of three nutritional regimes: CON fed from d 30 to 190 (n = 8), or NR/C (n = 7) or C/NR (n = 7) fed either the CON or NR diet from d 30 to 110 followed by CON or NR from d 110 to 190 of gestation. Cows were harvested on d 190 of gestation, and blood samples, fetal weights, and fetal tissue weights and samples were collected. Pancreas samples were embedded in paraffin and sectioned for standard immunohistochemistry procedures to quantify insulin-positive ß cells and number of apoptotic ß cells using TUNEL staining. Data were analyzed via ANOVA using the general linear model procedure of SAS. At harvest, empty carcass weights were decreased (P = 0.036) in fetuses of C/NR and NR/C fed dams compared to fetuses of CON fed dams. Pancreas weight was decreased (P = 0.028) in fetuses of C/NR fed dams compared to CON fetuses; however, fetuses of NR/C fed dams were not different (P > 0.05) from fetuses of CON fed dams. Maternal and fetal serum insulin concentrations were not different (P > 0.05) in NR/C fed compared to CON fed; however, concentrations of insulin were decreased (P = 0.036 and P = 0.40, respectively) in C/NR fed compared to CON fed. Beta cell number was decreased (P = 0.009) in fetuses of NR/C and C/NR fed dams compared to fetuses of CON fed dams. Percentage of apoptotic cells was increased (P < 0.0001) in fetuses of NR/C and C/NR fetuses than fetuses of CON fed dams. This evidence suggests that nutrient restriction either during early- or mid-gestation can negatively impact fetal pancreatic development. However, mid-gestational nutritional insult is potentially recovered by reacclimation to a diet that meets requirements of the dam, thus reducing negative outcomes in fetal offspring.

Effects of maternal nutrient restriction during early or mid-gestation without realimentation on maternal physiology and foetal growth and development in beef cattle.

The aim of this study is to determine the effects of early and mid-gestation nutrient restriction on maternal metabolites and foetal growth. Primiparous Angus cows were synchronized and inseminated with semen from one sire. Dietary treatments were: control to gain 1 kg/week (CON) or 0.55% maintenance energy and CP requirements (nutrient restricted; NR). A subset of dams was fed NR (n=8) or CON (n=8) from days 30 to 110 of gestation. Another group was fed CON (n=8), days 30 to 190; NR (n=7), days 30 to 110 followed by CON days 110 to 190; or CON, (n=7) days 30 to 110 followed by NR days 110 to 190. Cows were harvested at days 110 or 190 of gestation, when foetal measurements and samples were collected. Cows that were NR during days 30 to 110 or 110 to 190 of gestation lost significant BW and body condition score (P<0.001), this was associated with reduced plasma glucose during NR (P<0.002). Foetal weights, empty foetal weights, abdominal and thoracic circumferences were all reduced (P<0.03) in day 110 NR animals. Foetal perirenal adipose as a percentage of empty foetal weight was increased (P=0.01) in NR day 110 female foetuses compared with CON foetus. Maternal serum triglycerides at day 110 of gestation were decreased (P<0.05) in NR dams, whereas foetal serum triglycerides were increased (P<0.05) in response to maternal NR. Foetal weights tended to be reduced (P=0.08) in NR/CON and CON/NR v. CON/CON cattle at day 190 of gestation. Empty foetal weights, abdominal and thoracic circumferences were reduced (P⩽0.03) in NR/CON and CON/NR v. CON/CON cattle. Brain weight as a percentage of empty foetal weight was increased (P<0.001) in NR/CON and CON/NR v. CON/CON cattle. Foetal perirenal adipose as a percentage of empty foetal weight was increased (P=0.003) in NR/CON and CON/NR v. CON/CON cattle. Maternal serum triglycerides at day 190 of gestation were decreased (P<0.05) in association with maternal NR. Foetal serum triglycerides at day 190 of gestation were increased (P<0.05) in response to maternal NR during early gestation but decreased by NR in mid gestation compared with CON foetuses. The data show that maternal nutrient restriction during early or mid-gestation cause's asymmetrical foetal growth restriction, regardless if the restriction is preceded or followed by a period of non-restriction.

The effects of late gestation maternal nutrient restriction with or without protein supplementation on endocrine regulation of newborn and postnatal beef calves.

A study was conducted to evaluate late gestation maternal nutrient restriction (NR) with or without protein supplementation on endocrine regulation in newborn beef calves. This study used multiparous cows (4 and 5 years of age, n = 57) randomly assigned to one of three treatments for the last 100 days of gestation. The control (Con; n = 19) cows were fed to increase body condition score, whereas the NR (n = 19) and NR with protein supplement (NRS, n = 19) cows were fed to lose 1.2 ± 0.2 body condition score units during the last 100 days of gestation. Control cows were allowed ad libitum access to tall fescue/crabgrass paddock and, when grazing became insufficient, ad libitum hay was provided along with 1.3 kg of corn gluten feed 5 days/wk. Tall fescue paddocks were strip grazed to limit forage availability for NR and NRS. The NRS-treated dams were individually penned and fed 0.45 kg of soybean meal 3 days/wk. As forage became limited, the nutrient-restricted paddocks received limited fescue hay. After parturition cow/calf pairs were moved to a common pasture and received ad libitum silage and high-concentrate feed. Maternal NR regardless of supplementation reduced cow plasma glucose and insulin concentrations during late gestation (P < 0.0001 and P = 0.0051, respectively). Calves from NR dams weighed less at birth than Con calves (P = 0.04), whereas NRS calves were intermediate (33.4 ± 1.2, 35.0 ± 1.3, and 37.2 ± 1.3 kg NR, NRS, and Con, respectively). Plasma glucose concentrations of unsuckled calves at birth were reduced (P = 0.037) in NR and NRS calves compared with Con (67.7 ± 6.5 and 60.1 ± 6.9 vs. 83.7 ± 6.1 mg/dL, respectively). At birth, Con and NRS calves had increased (P = 0.0037) plasma leptin concentrations compared with NR calves, whereas calf plasma cortisol concentrations were greater for the nutrient-restricted groups than the Con group (treatment × day P = 0.0135). Plasma IgG concentrations from calves at 5 days of age were similar (P = 0.701) between maternal late gestation treatments. This research reports that late gestation NR reduces postnatal calf birth weight, plasma glucose and leads to reduced plasma leptin. Maternal protein supplementation appears to partially alleviate the effects of late gestation NR on reducing plasma leptin, birth weight, and growth rate from Day 30 of age to weaning.

Real-Time PCR Assays for the Detection of Puccinia psidii.

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and ß-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Identification, Virulence, and Distribution of Two Biovars of Pseudomonas syringae pv. actinidiae in New Zealand.

Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, was detected for the first time in New Zealand in November 2010. Only in Bay of Plenty, one of the four regions where this pathogen had been detected, did symptoms evolve beyond leaf spots, resulting in cane die-back, wilting of canes, and canker, sometimes leading to death of the vine. Molecular analysis (cts haplotype and BOX-polymerase chain reaction [PCR] electrophoretic pattern) of strains isolated from different regions of New Zealand revealed that two biovars could be distinguished. They have been called biovar 3 and biovar 4 to differentiate them from strains from Japan (biovar 1) or Korea (biovar 2), which have a different cts haplotype or a different BOX-PCR pattern. Biovars 3 and 4 displayed different degrees of virulence, as measured by their ability to cause leaf spots on young, potted kiwifruit plants. Biovar 3, which has also been present in Italy since 2008 and in France, was found in the Bay of Plenty, where cane diebacks were observed. In contrast, no symptoms other than leaf spots have been observed in orchards where strains of biovar 4 have been isolated. We report the distribution and the disease progression of biovars 3 and 4 in New Zealand.

Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae.

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.

Cold storage affects survival and growth of Erwinia amylovora on the calyx of apple.

AIMS: To determine the effect of cold storage on the survival of Erwinia amylovora. METHODS AND RESULTS: The survival of E. amylovora was assessed during storage at 2 degrees C. Populations of E. amylovora inoculated into phosphate-buffered saline remained static, whereas in nutrient media populations increased at low temperatures. In contrast, populations of E. amylovora on tissue in the apple calyx decreased during cold storage. CONCLUSIONS: Erwinia amylovora has the ability, in nutrient media, to multiply at low temperatures. However, populations of E. amylovora on tissue in the apple calyx decrease with the time spent in cold storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold storage of apples will provide assurance that mature fruit from orchards, free of fire blight, or even with low levels of fire blight, may be exported with a negligible risk of introducing the disease into countries free of fire blight.

Anti-class II monoclonal antibody-targeted Vibrio cholerae TcpA pilin: modulation of serologic response, epitope specificity, and isotype.

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.

Evaluation of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice.

Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD(50)) challenge and 50% protection against a 10-LD(50) challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.

Molecular and morphological reassessment of relationships within the Vittadinia group of Astereae (Asteraceae).

Morphological and ITS (internal transcribed spacer) sequence data for 40 species of the Austral-Pacific genera Camptacra, Kippistia, Minuria, Peripleura, Tetramolopium, and Vittadinia as well as one semiherbaceous species of Olearia were subjected to cladistic analysis, separately and together. Minuria, Peripleura, and Tetramolopium are paraphyletic as currently defined. Tetramolopium vagans from Australia appears to represent an undescribed genus. Both Kippistia suadefolia and Peripleura diffusa show close affinity to Minuria species, and Minuria macrorhiza appears to contain two distinct but closely related species. Vittadinia and the remaining species of Tetramolopium and Peripleura form a strong affinity group. The distribution of indels and the combined analysis each provide evidence that the Hawaiian and Cook Island species of Tetramolopium are descended from New Guinea species. The combined analysis also suggests that the Cook Island species T. mitiaroense is sister to the Hawaiian clade. Olearia arguta groups strongly with Camptacra and shows no close affinity with either of the arborescent species of Olearia used to root these analyses. Marked homoplasy among morphological characters indicates why generic delimitation in the group has been problematic.

The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton.

The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. The V. cholerae O1 El Tor biotype and V. cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V. cholerae O1 classical biotype strains do not. Although V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. Since V. cholerae O1 El Tor and V. cholerae O139 have displaced V. cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation of mshA in V. cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating adherence. Adherence of the V. cholerae O1 classical mshA mutant was unaffected. The results suggest that MSHA is a factor contributing to the ability of V. cholerae to adhere to plankton. The results also showed that both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.

Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade.

H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae.

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus-pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.

The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases.

Type 4 prepilins or prepilin-like-proteins are secreted by a wide range of bacterial species and are required for a variety of functions including type 4 pilus formation, toxin and other enzyme secretion, gene transfer, and biofilm formation. A distinctive feature of these proteins is the presence of a specialized leader peptide that is cleaved off by a cognate membrane-bound type 4 prepilin peptidase (TFPP) during the process of secretion. In this report we show that the TFPPs represent a novel family of bilobed aspartate proteases that is unlike any other protease. The active site pairs of aspartic acids of the two TFPPs in Vibrio cholerae are found at positions 125 and 189 of TcpJ and 147 and 212 of VcpD. Corresponding aspartate residues are completely conserved throughout this extensive peptidase family.

Differential transcription of the tcpPH operon confers biotype-specific control of the Vibrio cholerae ToxR virulence regulon.

Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30 degrees C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of toxT transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal toxT transcription. Transcription of tcpPH in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of toxT. In this study, we show a near absence of tcpPH message in the El Tor biotype under ToxR-inducing conditions of temperature and pH. However, once expressed, El Tor TcpP and TcpH appear to be as effective as classical TcpP and TcpH in activating toxT transcription. These results suggest that differences in regulation of virulence gene expression between the biotypes of V. cholerae primarily result from differences in expression of tcpPH message in response to environmental signals. We present an updated model for control of the ToxR virulence regulon in V. cholerae.

A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon.

The expression of the ToxR virulence regulon is dependent upon the regulatory proteins ToxR/ToxS, TcpP/TcpH and ToxT. We describe here a previously unidentified gene in Vibrio cholerae, aphA (activator of tcpP and tcpH expression), which is required for the transcription of the tcpPH operon. Under conditions normally optimal for virulence gene expression, an in frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx-lacZ) and prevented the production of the toxin co-regulated pilus (TCP). Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx-lacZ expression and TCP production in the delta aphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcription of tcpPH. Indeed, the expression of a chromosomal tcpP-lacZ fusion was reduced in the delta aphA mutant and increased in both V. cholerae and Escherichia coli by introducing aphA expressed from an inducible promoter. These results support a model in which AphA functions at a previously unknown step in the ToxR virulence cascade to activate the transcription of tcpPH. TcpP/TcpH, together with ToxR/ToxS, then activate the expression of toxT, resulting ultimately in the production of virulence factors such as cholera toxin and TCP.

Genetic and transcriptional analyses of the Vibrio cholerae mannose-sensitive hemagglutinin type 4 pilus gene locus.

The mannose-sensitive hemagglutinin (MSHA) of the Vibrio cholerae O1 El Tor biotype is a member of the family of type 4 pili. Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer. The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the V. cholerae chromosome. Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified. Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes. This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci. The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB of Escherichia coli. In E. coli, the yhdA and mreB genes are adjacent to each other on the chromosome. The finding that the MSHA locus lies between these two E. coli homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.