Kegg Pathway: Cholera - Colonization

Infect Immun. 2009 Oct 26;
Zhang W, Zhang C, Francis DH, Fang Y, Knudsen D, Nataro JP, Robertson DC

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea disease in humans and farm animals. E. coli fimbriae or Colonization factor antigens (CFAs) and enterotoxins including heat-labile (LT) and heat-stable (ST) are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not been much included as an antigen in vaccine development against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated porcine LT (pLT) gene for a pLT192(R-->G) toxoid and STa (pSTa) gene for three full-length pSTatoxoids [STa11(N-->K), STa12(P-->F), and STa13(A-->Q)], and used the full-length pLT192 as an adjuvant to carry the pSTatoxoid for 'pLT192:pSTa-toxoid' fusion antigens. Rabbits immunized with 'pLT192:pSTa12' or 'pLT192:pSTa13' fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified Cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born from a sow immunized with the 'pLT192:pSTa13' fusion antigen were protected when challenged with a STa-positive ETEC. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid, and elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC associated diarrhea in animals and humans.

Curr Top Microbiol Immunol. 2009; 337: 37-59
Ritchie JM, Waldor MK

Vibrio Cholerae is a curved Gram-negative rod that causes the diarrheal disease Cholera. One hundred and twenty five years of study of V. Cholerae microbiology have made this lethal pathogen arguably the most well-understood non-invasive mucosal pathogen. Over the past 25 years, modern molecular techniques have permitted the identification of many genes and cellular processes that are critical for V. Cholerae Colonization of the gastrointestinal tract. Review of the literature reveals that there are two classes of genes that influence V. Cholerae Colonization of the suckling mouse intestine, the most commonly used animal model to study V. Cholerae pathogenesis. Inactivation of one class of genes results in profound attenuation of V. Cholerae intestinal Colonization, whereas inactivation of the other class of genes results in only moderate Colonization defects. The latter class of genes suggests that V. Cholerae may colonize several intestinal niches that impose distinct requirements and biological challenges, thus raising the possibility that there is physiologic heterogeneity among the infecting population. Efficient V. Cholerae intestinal Colonization and subsequent dissemination to the environment appears to require temporally ordered expression of sets of genes during the course of infection. Key challenges for future investigations of V. Cholerae pathogenicity will be to assess the degree of heterogeneity in the infecting population, whether such heterogeneity has functional significance, and if stochastic processes contribute to generation of heterogeneity in vivo.

PLoS One. 2009; 4(10): e7352
Olivier V, Queen J, Satchell KJ

Vibrio Cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this Colonization were investigated. Successful Colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for Colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. Cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once Colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause Cholera disease. This adult mouse model of intestinal V. Cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

Microbes Infect. 2009 Sep 24;
Hong W, Peng D, Rivera M, Gu XX

Otitis media (OM) can occur following outset of upper respiratory tract infections. Inhibition of bacterial Colonization in nasopharynx (NP) by mucosal vaccination may prevent OM by reducing bacterial invasion of the middle ears (MEs). In this study, 80 chinchillas were intranasally (i.n.) immunized with a detoxified lipooligosaccharide (dLOS)-tetanus toxoid conjugate vaccine of nontypeable Haemophilus influenzae (NTHi) mixed with Cholera toxin (CT) or CT alone. All vaccinated animals responded with elevated levels of mucosal and serum anti-LOS antibodies. Two weeks after the last immunization, 40 chinchillas were challenged i.n. with NTHi to evaluate NP Colonization and ME infection while the rest of the animals were challenged transbullarly (T.B.) to examine the development of OM. Compared to the control group, the vaccination inhibited not only bacterial Colonization in NP and transmission to MEs in the i.n. challenge group but also bacterial Colonization in NP and transmission to unchallenged ears in the T.B. challenge group. Though no difference was found in the challenged ears of either group right after the T.B. challenge, an early clearance of NTHi from NP and unchallenged ears as well as less severity of OM in the unchallenged ears were observed in vaccinated animals. Current results along with our previous data indicate that mucosal vaccination is capable of inhibiting NTHi NP Colonization and preventing OM occurrence in chinchillas; the i.n. challenge model is preferable for testing the mucosal vaccines while the T.B. challenge model is superior for testing the systemic vaccines.

J Bacteriol. 2009 Nov; 191(22): 7027-38
Moisi M, Jenul C, Butler SM, New A, Tutz S, Reidl J, Klose KE, Camilli A, Schild S

The facultative pathogen Vibrio Cholerae is the causative agent of the human intestinal disease Cholera. Both motility and chemotaxis of V. Cholerae have been shown to contribute to the virulence and spread of Cholera. The flagellar gene operons are organized into a hierarchy composed of four classes (I to IV) based on their temporal expression patterns. Some regulatory elements involved in flagellar gene expression have been elucidated, but regulation is complex and flagellar biogenesis in V. Cholerae is not completely understood. In this study, we determined that the virulence defect of a V. Cholerae cheW1 deletion mutant was due to polar effects on the downstream open reading frame VC2058 (flrD). Expression of flrD in trans restored the virulence defect of the cheW1 deletion mutant, and deletion of flrD resulted in a V. Cholerae strain attenuated for virulence, as determined by using the infant mouse intestinal Colonization model. The flrD mutant strain exhibited decreased transcription of class III and IV flagellar genes and reduced motility. Transcription of the flrD promoter, which lies within the coding sequence of cheW1, is independent of the flagellar transcriptional activators FlrA and RpoN, which activate class II genes, indicating that flrD does not fit into any of the four flagellar gene classes. Genetic epistasis studies revealed that the two-component system FlrBC, which is required for class III and IV flagellar gene transcription, acts downstream of flrD. We hypothesize that the inner membrane protein FlrD interacts with the cytoplasmic FlrBC complex to activate class III and IV gene transcription.

Infect Immun. 2009 Dec; 77(12): 5206-15
Glenn GM, Francis DH, Danielsen EM

Recent studies have confirmed older observations that the enterotoxins enhance enteric bacterial Colonization and pathogenicity. How and why this happens remains unknown at this time. It appears that toxins such as the heat-labile enterotoxin (LT) from Escherichia coli can help overcome the innate mucosal barrier as a key step in enteric pathogen survival. We review key observations relevant to the roles of LT and Cholera toxin in protective immunity and the effects of these toxins on innate mucosal defenses. We suggest either that toxin-mediated fluid secretion mechanically disrupts the mucus layer or that toxins interfere with innate mucosal defenses by other means. Such a breach gives pathogens access to the enterocyte, leading to binding and pathogenicity by enterotoxigenic E. coli (ETEC) and other organisms. Given the common exposure to LT(+) ETEC by humans visiting or residing in regions of endemicity, barrier disruption should frequently render the gut vulnerable to ETEC and other enteric infections. Conversely, toxin immunity would be expected to block this process by protecting the innate mucosal barrier. Years ago, Peltola et al. (Lancet 338:1285-1289, 1991) observed unexpectedly broad protective effects against LT(+) ETEC and mixed infections when using a toxin-based enteric vaccine. If toxins truly exert barrier-disruptive effects as a key step in pathogenesis, then a return to classic toxin-based vaccine strategies for enteric disease is warranted and can be expected to have unexpectedly broad protective effects.

J Bacteriol. 2009 Nov; 191(22): 6911-7
Jude BA, Martinez RM, Skorupski K, Taylor RK

Vibrio Cholerae is the etiologic agent of Cholera in humans. Intestinal Colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and Cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. Cholerae. HapR represses the expression of genes encoding regulators involved in V. Cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, Delta hapA Delta prtV strains attach to chitin beads more efficiently than either the WT or a Delta hapA Delta prtV Delta gbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.

J Bacteriol. 2009 Nov; 191(21): 6555-70
Syed KA, Beyhan S, Correa N, Queen J, Liu J, Peng F, Satchell KJ, Yildiz F, Klose KE

Vibrio Cholerae is a motile bacterium responsible for the disease Cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. Cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal Colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates Colonization within the intestine and repression of virulence factors maximally induced following Colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. Cholerae Colonization and disease progression.

Microbiol Immunol. 2009 Apr; 53(4): 233-40
Scavone P, Rial A, Umpierrez A, Chabalgoity A, Zunino P

Proteus mirabilis is commonly associated with complicated UTI and expresses several virulence factors, including MR/P fimbriae. In the present study mice were immunised nasally with MrpA, the structural subunit of MR/P, with or without CT as a mucosal adjuvant. The animals were then challenged with P. mirabilis and induction of specific serum and urine IgG and IgA, IFN-gamma production and bacterial kidney and bladder Colonization were assessed. MrpA-immunised mice exhibited significant induction of serum IgA and urine IgA and IgG. MrpA/CT-immunised mice showed both significant serum and urine IgA and IgG production. Only this group showed significant IFN-y production. Both groups of animals had significant decrease in bacterial Colonization of kidneys but not of bladders. No correlation between specific antibody induction in serum and CFU decrease was observed in any group of animals. Our results suggest that a mucosal adjuvant (CT) in the urinary tract enhanced humoral and cytokine response although it did not influence the degree of protection against UTI provided by MrpA. Further studies are necessary to understand immune modulation in the urinary tract.

Commun Integr Biol. 2008 Jul; 1(1): 42-4
Tsou AM, Frey EM, Hsiao A, Liu Z, Zhu J

Pathogenic bacteria, such as Vibrio Cholerae, must be capable of adapting to diverse living conditions, especially when transitioning from life in environmental reservoirs to life in a host. The abilities to sense arrival at a site suitable for Colonization or infection and to respond with appropriate alterations in gene expression are crucial for a pathogen's success. Recently, we have shown that V. Cholerae is able to recognize that it has reached its Colonization site in the small intestine by sensing breakage of its flagellum as it penetrates the mucosal layer overlaying the intestinal epithelium. Flagellar loss results in the release of the anti-sigma factor FlgM and subsequent activation of the alternative sigma-factor FliA. FliA represses the quorum sensing-controlled transcriptional regulator, HapR, allowing increased expression of virulence factors such as Cholera Toxin (CT) and the Toxin Coregulated Pilus (TCP). In this way, the de-repression of virulence factor expression coincides with the arrival of bacteria at the site of infection at the intestinal mucosa. Our work reveals an interesting interplay between motility and quorum sensing signaling pathways to precisely time virulence gene expression during Colonization.

FEMS Immunol Med Microbiol. 2009 Oct; 57(1): 69-79
Morin CE, Kaper JB

Live, attenuated Vibrio Cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. Cholerae strains must be created that stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo-induced promoters have been shown to increase the stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo-induced promoters: V. Cholerae P(argC), P(fhuC) and P(vca1008), and Salmonella enterica serovar Typhi P(ompC). We demonstrate that several of these expression plasmids meet two critical criteria for V. Cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. Cholerae vaccine strain CVD 103-HgR at low copy number, in the absence of selective pressure. Second, real-time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. Cholerae Colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various time points.

J Bacteriol. 2009 Sep; 191(18): 5669-79
Martinez RM, Dharmasena MN, Kirn TJ, Taylor RK

Vibrio Cholerae is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of V. Cholerae, demonstrating that motility is an important virulence factor. FlrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 (flgP) and VC2207 (flgO) were identified as being regulated by FlrC via a microarray analysis of an flrC mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a Colonization factor. The study reported here focuses on the characterization of flgO, the first gene in the flgOP operon. We show that FlgO and FlgP are important for motility, as strains with mutations in the flgOP genes have reduced motility phenotypes. The flgO and flgP mutant populations display fewer motile cells as well as reduced numbers of flagellated cells. The flagella produced by the flgO and flgP mutant strains are shorter in length than the wild-type flagella, which can be restored by inhibiting rotation of the flagellum. FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus Colonization by V. Cholerae O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FlgP function in motility to mediate flagellar stability and influence attachment and Colonization.

BMC Microbiol. 2009; 9: 114
Zhang L, Zhu Z, Jing H, Zhang J, Xiong Y, Yan M, Gao S, Wu LF, Xu J, Kan B

BACKGROUND: The Twin-arginine translocation (Tat) system serves to translocate folded proteins, including periplasmic enzymes that bind redox cofactors in bacteria. The Tat system is also a determinant of virulence in some pathogenic bacteria, related to pleiotropic effects including growth, motility, and the secretion of some virulent factors. The contribution of the Tat pathway to Vibrio Cholerae has not been explored. Here we investigated the functionality of the Tat system in V. Cholerae, the etiologic agent of Cholera. RESULTS: In V. Cholerae, the tatABC genes function in the translocation of TMAO reductase. Deletion of the tatABC genes led to a significant decrease in biofilm formation, the ability to attach to HT-29 cells, and the ability to colonize suckling mouse intestines. In addition, we observed a reduction in the output of Cholera toxin, which may be due to the decreased transcription level of the toxin gene in tatABC mutants, suggesting an indirect effect of the mutation on toxin production. No obvious differences in flagellum biosynthesis and motility were found between the tatABC mutant and the parental strain, showing a variable effect of Tat in different bacteria. CONCLUSION: The Tat system contributes to the survival of V. Cholerae in the environment and in vivo, and it may be associated with its virulence.

Infect Immun. 2009 Aug; 77(8): 3475-84
Nygren E, Li BL, Holmgren J, Attridge SR

We describe here a new animal model that offers the prospect of using conventional adult mice for direct evaluation of the protective potential of new Cholera vaccines. Pretreatment of adult mice with oral streptomycin allowed intestinal Colonization by streptomycin-resistant Vibrio Cholerae strains of either the O1 or the O139 serogroup. Bacteria were recovered in greatest numbers from the cecum and large intestine, but recoveries from all regions of the gut correlated significantly with bacterial excretion in fresh fecal pellets, which thus provides a convenient indicator of the extent and duration of gut Colonization. Mice immunized mucosally or systemically with viable or inactivated V. Cholerae were shown to be comparatively refractory to Colonization after challenge with the immunizing strain. Several variables were examined to optimize the model, the most significant being the size of the challenge inoculum; surprisingly, a smaller challenge dose resulted in more consistent and sustained Colonization. Studies with mutant strains unable to produce Cholera toxin or toxin-coregulated pili revealed that neither factor contributed significantly to Colonization potential. Protection against V. Cholerae challenge was shown to be serogroup restricted, and significant inverse correlations were detected between serum and intestinal anti-lipopolysaccharide antibody responses and the levels of excretion of challenge organisms.

J Bacteriol. 2009 Jun; 191(11): 3665-76
Krebs SJ, Kirn TJ, Taylor RK

Colonization of the human small intestine by Vibrio Cholerae requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the tcp operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for Colonization. Bacteria lacking tcpF are deficient in Colonization, and anti-TcpF antibodies are protective in the infant mouse Cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in Colonization, random mutagenesis of tcpF was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence extracellular secretion and that determinants near the C terminus are important for the function of TcpF in Colonization. The TcpF proteins of certain environmental V. Cholerae isolates with 31% to 66% identity to pathogenic V. Cholerae TcpF showed higher similarity in regions identified as secretion determinants but diverged in regions found to be important for Colonization. These environmental TcpF proteins are secreted from the pathogenic strain; however, they do not mediate Colonization in the infant mouse model. Here we provide genetic evidence pointing toward regions of TcpF that influence secretion, as well as regions that play an important role in in vivo Colonization.

Infect Immun. 2009 May; 77(5): 2076-83
Lu YJ, Forte S, Thompson CM, Anderson PW, Malley R

Cell wall polysaccharide (CWPS), pneumolysin, and surface adhesin A (PsaA) are antigens common to virtually all serotypes of Streptococcus pneumoniae (pneumococcus), and all have been studied separately for use in protection. Previously we showed that protection against nasopharyngeal (NP) Colonization by intranasal vaccination of mice with killed pneumococci is mediated by T(H)17 cells and correlates with interleukin-17A (IL-17A) expression by T cells in vitro; we have also shown that CWPS and other species-common antigens protect against Colonization by a similar mechanism. Here we made a fusion protein of PsaA with the pneumolysin nontoxic derivative PdT and then coupled CWPS to the fusion protein, aiming to enhance immune responses to all three antigens. When given intranasally with Cholera toxin adjuvant, the fusion conjugate induced higher serum antibody titers and greater priming for IL-17A responses than an equimolar mixture of the three antigens. The conjugate administered intranasally protected mice against experimental NP Colonization by a strain of serotype 6B, while mice immunized with the mixture or with bivalent conjugates were not protected. Subcutaneous immunization with the conjugate and alum adjuvant likewise induced higher antibody titers than the mixture, primed for IL-17A responses, and reduced Colonization. The conjugate, but not the antigen mixture, fully protected mice from fatal pneumonia caused by a highly virulent serotype 3 strain. Thus, a covalent construct of three antigens common to all serotypes exhibits protection with both mucosal and systemic administration.

Infect Immun. 2009 May; 77(5): 2125-35
Ghosal A, Bhowmick R, Banerjee R, Ganguly S, Yamasaki S, Ramamurthy T, Hamabata T, Chatterjee NS

CS6 is a widely expressed Colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for Colonization.

Exp Biol Med (Maywood). 2009 Apr; 234(4): 403-9
Shah P, Briles DE, King J, Hale Y, Swiatlo E

Streptococcus pneumoniae is an encapsulated pathogen that can cause invasive disease following Colonization of the nasopharynx. Targeting Colonization of mucosal surfaces may, therefore, be the best approach for vaccination to prevent pneumococcal invasive disease. Previous studies in our laboratory have shown that immunization with recombinant polyamine transport protein D (PotD) protects mice against systemic pneumococcal infections. In this study we investigated the efficacy of mucosal immunization with rPotD to protect against pneumococcal carriage and invasion in a murine model. Mice were intranasally immunized with either rPotD and Cholera toxin B subunit (CTB) or CTB alone. Significantly less pneumococci were recovered from the nasopharynx of immunized mice compared to the control animals following intranasal challenge with either EF3030 (serotype 19F) (P < 0.05) or an invasive serotype 4 isolate (TIGR4) (P < 0.05). PotD immunized mice also had lesser bacteria in their sinus tissues (P < 0.05), brains (P < 0.05), lungs and olfactory bulbs following intranasal challenge with TIGR4. ELISA analysis demonstrated the presence of IgG antibodies to PotD in the serum and IgA antibodies in the saliva. These results indicate that mucosal immunization with PotD generates both mucosal and systemic immune responses and prevents establishment of nasopharyngeal carriage by multiple pneumococcal serotypes. Thus, PotD is a potentially important antigen for development of a pneumococcal protein vaccine.

Infect Immun. 2009 Apr; 77(4): 1613-22
Bogaert D, Weinberger D, Thompson C, Lipsitch M, Malley R

Streptococcus pneumoniae Colonization and invasive disease peak around the third and first birthdays, respectively, and decline thereafter. While these declines are attributable in part to immunity acquired via natural exposure, maturation of innate immune responses may also be involved. A mucosally administered candidate whole-cell pneumococcal vaccine (WCV) containing killed pneumococcal antigen (WCA) plus a Cholera toxin adjuvant protects against intranasal carriage of pneumococci by a mechanism that is antibody independent and CD4(+) TH17 cell dependent. Because infants and children are a key target population for this vaccine, we sought to evaluate the immune responses of neonatal and infant mice to S. pneumoniae and to assess whether the WCV would be effective in these mice. Like human infants, infant mice showed impaired clearance of nasopharyngeal Colonization with S. pneumoniae. Macrophages from neonatal and infant mice stimulated with killed pneumococci in vitro showed significantly reduced cytokine production, including that of KC, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage chemoattractant protein 1, interleukin-6 (IL-6), IL-1alpha, tumor necrosis factor alpha, and gamma interferon, whereas IL-10 expression was significantly increased compared to that in macrophages from adult mice. IL-17A production from adult immune CD4(+) T cells was significantly delayed when neonatal macrophages instead of adult macrophages were used as antigen-presenting cells. Moreover, whole blood from mice immunized as neonates with WCV produced significantly less IL-17A after stimulation with WCA than did blood from mice immunized as adults. Nonetheless, a single immunization of neonatal mice with WCV significantly reduced Colonization density. Overall, our data suggest an impairment of both innate and acquired cellular immune responses in neonatal and infant mice. However, WCV confers a significant reduction in Colonization following pneumococcal challenge, suggesting that it may still be effective in the setting of immature immune responses.

J Clin Microbiol. 2009 Apr; 47(4): 1087-95
Chatterjee S, Ghosh K, Raychoudhuri A, Chowdhury G, Bhattacharya MK, Mukhopadhyay AK, Ramamurthy T, Bhattacharya SK, Klose KE, Nandy RK

The incidence of Vibrio Cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% (n = 54) of the total 197 V. Cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of Cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher Colonization in an animal model compared to those lacking tcpA. PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA, rtxA, Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.