Search

A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

In the Gram-positive pathogen Staphylococcus aureus, pore-forming toxins (PFTs), such as leukocidins and hemolysins, play prominent roles in staphylococcal pathogenesis by killing host immune cells and red blood cells (RBCs). However, it remains unknown which combination of toxin antigens would induce the broadest protective immune response against those toxins. In this study, by targeting six major staphylococcal PFTs (i.e., gamma-hemolysin AB [HlgAB], gamma-hemolysin CB [HlgCB], leukocidin AB [LukAB], leukocidin ED [LukED], Panton-Valentine leukocidin [LukSF-PV], and alpha-hemolysin [Hla]), we generated 10 recombinant toxins or toxin subunits, 3 toxoids, and their rabbit antibodies. Using the cytolytic assay for RBCs and polymorphonuclear cells (PMNs), we determined the best combination of toxin antibodies conferring the broadest protection against those staphylococcal PFTs. Although anti-HlgA IgG (HlgA-IgG) showed low cross-reactivity to other toxin components, it was essential to protect rabbit and human RBCs and human PMNs. For the protection of rabbit RBCs, Hla(H35L) toxoid-IgG was also required, whereas for human PMNs, LukS-IgG and LukA(E323A)B-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, Hla(H35L), and LukA(E323A)B were used to immunize rabbits, they increased rabbit survival; however, they did not block staphylococcal abscess formation in kidneys. Based on these results, we proposed that the combination of HlgA, LukS, Hla(H35L), and LukA(E323A)B is the optimal vaccine component to protect human RBCs and PMNs from staphylococcal PFTs. We also concluded that a successful S. aureus vaccine requires not only those toxin antigens but also other antigens that can induce immune responses blocking staphylococcal colonization.

In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.

Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi. No vaccine is available for humans. Dogmatically, B. burgdorferi can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the B. burgdorferi spirochete is effectively cleared by anti-B. burgdorferi antibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous B. burgdorferi spirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S. Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA. We determined that S. Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S. Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (10(9) CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD(50) (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S. Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo . Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage.

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro , including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis (IE), particularly in people with predisposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. In this study, we compared the genomes of a collection of S. sanguinis strains comprised of both oral isolates and bloodstream isolates from patients diagnosed with IE. Oral and IE isolates could not be distinguished by phylogenetic analyses, and we did not succeed in identifying virulence genes unique to the IE strains. We then investigated the virulence of these strains in a rabbit model of IE using a variation of the Bar-seq (barcode sequencing) method wherein we pooled the strains and used Illumina sequencing to count unique barcodes that had been inserted into each isolate at a conserved intergenic region. After we determined that several of the genome sequences were misidentified in GenBank, our virulence results were used to inform our bioinformatic analyses, identifying genes that may explain the heterogeneity in virulence. We further characterized these strains by assaying for phenotypes potentially contributing to virulence. Neither strain competition via bacteriocin production nor biofilm formation showed any apparent relationship with virulence. Increased cell-associated manganese was, however, correlated with blood isolates. These results, combined with additional phenotypic assays, suggest that S. sanguinis virulence is highly variable and results from multiple genetic factors.

Plasmodium vivax parasites preferentially invade reticulocytes in human beings. P. vivax merozoite surface protein 1 (PvMSP1) and PvMSP1 paralog (PvMSP1P) may have important functions in reticulocyte adherence during invasion. These proteins share similar structures, including the presence of two epidermal growth factor (EGF)-like and glycosylphosphatidylinositol (GPI)-anchored domains at the C terminus. However, there have been no reports concerning the functional activity of PvMSP1P in reticulocyte adherence during P. vivax invasion. In this study, the ability of PvMSP1P-19 to bind to reticulocytes and normocytes was analyzed. The reticulocyte binding activity of PvMSP1P-19 was 4.0-fold higher than its normocyte binding activity. The binding of PvMSP1P-19 to reticulocytes and normocytes was inhibited in a dose-dependent manner by antibodies from immunized rabbits and by antibodies from vivax parasite-infected patients. Consistently, antibodies against PvMSP1P inhibited parasite invasion during short-term in vitro cultivation. Similar to the case for PvDBPII binding activity, PvMSP1P-19 binding activity was reduced in chymotrypsin-treated reticulocytes. However, no significant difference between the binding of PvMSP1P-19 to Duffy-positive and Duffy-negative erythrocytes was found. The minimal binding motif of PvMSP1P-19 was characterized using synthetic peptides. The results showed that the residues at amino acid positions 1791 to 1808 may have an important function in mediating merozoite adherence to reticulocytes. The positively charged residues within the EGF-like domain were shown to constitute a key binding motif. This work presents strong evidence supporting the role of PvMSP1P in host target cell selection and invasion of Duffy-independent pathway in P. vivax. Moreover, PvMSP1P-19-specific antibodies may confer protection against P. vivax reinvasion.

Coagulase activation of prothrombin by staphylococcus induces the formation of fibrin deposition that facilitates the establishment of infection by Staphylococcus species. Coagulase activity is a key characteristic of Staphylococcus pseudintermedius; however, no coagulase gene or associated protein has been studied to characterize this activity. We report a recombinant protein sharing 40% similarity to Staphylococcus aureus coagulase produced from a putative S. pseudintermedius coagulase gene. Prothrombin activation by the protein was measured with a chromogenic assay using thrombin tripeptide substrate. Stronger interaction with bovine prothrombin than with human prothrombin was observed. The S. pseudintermedius coagulase protein also bound complement C3 and immunoglobulin. Recombinant coagulase facilitated the escape of S. pseudintermedius from phagocytosis, presumably by forming a bridge between opsonizing antibody, complement, and fibrinogen. Evidence from this work suggests that S. pseudintermedius coagulase has multifunctional properties that contribute to immune evasion that likely plays an important role in virulence.

Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration., Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration. In this study, we report a novel protein that possesses a Trx domain-like structure of the thioredoxin family and is expressed on the surface of merozoites of the malaria parasite Plasmodium falciparum. This protein, namely, PfTrx-mero protein, displayed a mutated sequence character at the Trx domain, but with a specific binding activity to human erythrocytes. Specific antibodies to the protein inhibited merozoite invasion into human erythrocytes. Immunization with a homologous protein of Plasmodium berghei strain ANKA also showed significant protection against lethal infection in mice. These results suggested that the novel PfTrx-like-mero protein expressed on the surface of merozoites is an important ligand participating in erythrocyte invasion and a potential vaccine candidate.

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a chronic, sexually transmitted infection characterized by multiple symptomatic and asymptomatic stages. Although several other species in the genus are able to cause or contribute to disease, T. pallidum differs in that it is able to rapidly disseminate via the bloodstream to tissue sites distant from the site of initial infection. It is also the only Treponema species able to cross both the blood-brain and placental barriers. Previously, the T. pallidum proteins, Tp0750 and Tp0751 (also called pallilysin), were shown to degrade host proteins central to blood coagulation and basement membrane integrity, suggesting a role for these proteins in T. pallidum dissemination and tissue invasion. In the present study, we characterized Tp0750 and Tp0751 sequence variation in a diversity of pathogenic and nonpathogenic treponemes. We also determined the proteolytic potential of the orthologs from the less invasive species Treponema denticola and Treponema phagedenis . These analyses showed high levels of sequence similarity among Tp0750 orthologs from pathogenic species. For pallilysin, lower levels of sequence conservation were observed between this protein and orthologs from other treponemes, except for the ortholog from the highly invasive rabbit venereal syphilis-causing Treponema paraluiscuniculi . In vitro host component binding and degradation assays demonstrated that pallilysin and Tp0750 orthologs from the less invasive treponemes tested were not capable of binding or degrading host proteins. The results show that pallilysin and Tp0750 host protein binding and degradative capability is positively correlated with treponemal invasiveness.

Photobacterium damselae subsp. damselae , an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for “photobacterial lysin encoded on a plasmid.” PhlyP formed stable oligomers and small membrane pores, causing efflux of K + , with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence.

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum , the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis.

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a Plasmodium falciparum multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP119) to P. falciparum MSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP119. Here, using the PfMSP1/8 construct, we further optimized the recombinant PfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate Pfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of Pfs25 block sexual-stage development in mosquitoes. The sequence encoding mature Pfs25 was codon harmonized for expression in Escherichia coli. We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with the Pfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native Pfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of PfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli , refolded in vitro , and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 ( r claudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as r claudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo . Significant protection of Caco-2 cells was also observed using either r claudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since r claudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

In El Tor biotype strains of toxigenic Vibrio cholerae , the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC , the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.

Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

We previously showed that Brucella abortus rough mutant strain 2308 Δ ATP (called the Δ rfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the Δ rfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene ( asp24 ) expression was significantly upregulated in the Δ rfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE ( bab1_0542 ) and the permease gene rfbD ( bab1_0543 ), while the Δ wboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the Δ rfbE mutant by deleting the wboA gene (thereby creating the Δ rfbE Δ wboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the Δ rfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants.

Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats.

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens ). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease.

Francisella tularensis , a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 Δ guaBA , Δ aroD , and Δ fipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD 50 ). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The Δ guaBA and Δ aroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the Δ fipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The Δ guaBA and Δ aroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.

Atypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC . These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap . By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter- lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease.

Vibrio cholerae , an important human intestinal pathogen, is responsible for the diarrheal disease cholera. The pathogenesis of V. cholerae is a highly coordinated process that involves diverse regulatory factors. It has recently been demonstrated that disruption of the V. cholerae fadD gene, encoding a long-chain fatty acyl coenzyme A (acyl-CoA) ligase, drastically reduces expression of the major virulence genes and in vivo lethality of this important human pathogen. This effect was due to reduced membrane localization of the central virulence regulator TcpP. In this study, the reason for the impaired membrane localization of TcpP in the fadD mutant was investigated. We demonstrate that extracytoplasmic stress is induced in the V. cholerae Δ fadD strain. In response to the extracytoplasmic stress, the integral membrane protease RseP is activated and degrades the membrane-localized TcpP in the fadD mutant strain. Indeed, disruption of the rseP gene in a fadD mutant background restored membrane localization of TcpP and expression of the downstream virulence genes toxT , ctxA , and tcpA . Increased expression of the σ E regulon genes in ethanol-treated wild-type V. cholerae indicated that ethanol exposure could induce an extracytoplasmic stress response in V. cholerae . Ethanol treatment also led to activation of the RseP protease activity and resulted in degradation of membrane-localized TcpP and subsequent reduction in expression of the virulence genes. Taken together, these results suggest that extracytoplasmic stress response per se reduces virulence of V. cholerae by impairing membrane localization of TcpP.

Apical membrane antigen 1 (AMA1) is a leading vaccine candidate, but the allelic polymorphism is a stumbling block for vaccine development. We previously showed that a global set of AMA1 haplotypes could be grouped into six genetic populations. Using this information, six recombinant AMA1 proteins representing each population were produced. Rabbits were immunized with either a single recombinant AMA1 protein or mixtures of recombinant AMA1 proteins (mixtures of 4, 5, or 6 AMA1 proteins). Antibody levels were measured by enzyme-linked immunosorbent assay (ELISA), and purified IgG from each rabbit was used for growth inhibition assay (GIA) with 12 different clones of parasites (a total of 108 immunogen-parasite combinations). Levels of antibodies to all six AMA1 proteins were similar when the antibodies were tested against homologous antigens. When the percent inhibitions in GIA were plotted against the number of ELISA units measured with homologous AMA1, all data points followed a sigmoid curve, regardless of the immunogen. In homologous combinations, there were no differences in the percent inhibition between the single-allele and allele mixture groups. However, all allele mixture groups showed significantly higher percent inhibition than the single-allele groups in heterologous combinations. The 5-allele-mixture group showed significantly higher inhibition to heterologous parasites than the 4-allele-mixture group. On the other hand, there was no difference between the 5- and 6-allele-mixture groups. These data indicate that mixtures with a limited number of alleles may cover a majority of the parasite population. In addition, using the data from 72 immunogen-parasite combinations, we mathematically identified 13 amino acid polymorphic sites which significantly impact GIA activities. These results could be a foundation for the rational design of a future AMA1 vaccine.

In May 2011, a large food-borne outbreak was traced to an unusual O104:H4 enteroaggregative Escherichia coli (EAEC) strain that produced Shiga toxin (Stx) type 2 (Stx2). We developed a mouse model to study the pathogenesis and treatment for this strain and examined the virulence of the isolate for Dutch belted rabbits. O104:H4 strain C227-11 was gavaged into C57BL/6 mice at 10 9 to 10 11 CFU/animal. The infected animals were then given water with ampicillin (Amp; 5 g/liter) ad libitum . The C227-11-infected, Amp-treated C57BL/6 mice exhibited both morbidity and mortality. Kidneys from mice infected with C227-11 showed acute tubular necrosis, a finding seen in mice infected with typical Stx-producing E. coli . We provided anti-Stx2 antibody after infection and found that all of the antibody-treated mice gained more weight than untreated mice and, in another study, that all of the antibody-treated animals lived, whereas 3/8 phosphate-buffered saline-treated mice died. We further compared the pathogenesis of C227-11 with that of an Stx-negative (Stx − ) O104:H4 isolate, C734-09, and an Stx2 − phage-cured derivative of C227-11. Whereas C227-11-infected animals lost weight or gained less weight over the course of infection and died, mice infected with either of the Stx − isolates did not lose weight and only one mouse died. When the Stx-positive (Stx + ) and Stx2 − O104:H4 strains were compared in rabbits, greater morbidity and mortality were observed in rabbits infected with the Stx2 + isolates than the Stx2 − isolates. In conclusion, we describe two animal models for EAEC pathogenesis, and these studies show that Stx2 is responsible for most of the virulence observed in C227-11-infected mice and rabbits.

AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli , enteroaggregative E. coli , and Citrobacter rodentium . Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium . Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA . Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

Botulinum toxin is a highly potent oral and inhalation poison, which means that the toxin must have an efficient mechanism for penetration of epithelial barriers. To date, three models for toxin passage across epithelial barriers have been proposed: (i) the toxin itself undergoes binding and transcytosis; (ii) an auxiliary protein, HA35, transports toxin from the apical to the basal side of epithelial cells; and (iii) an auxiliary protein, HA35, acts on the basal side of epithelial cells to disrupt tight junctions, and this permits paracellular flux of toxin. These models were evaluated by studying toxin absorption following inhalation exposure in mice. Three types of experiments were conducted. In the first, the potency of pure neurotoxin was compared with that of progenitor toxin complex, which contains HA35. The results showed that the rate and extent of toxin absorption, as well as the potency of absorbed toxin, did not depend upon, nor were they enhanced by, the presence of HA35. In the second type of experiment, the potencies of pure neurotoxin and progenitor toxin complex were compared in the absence or presence of antibodies on the apical side of epithelial cells. Antibodies directed against the neurotoxin protected against challenge, but antibodies against HA35 did not. In the final type of experiment, the potency of pure neurotoxin and toxin complex was compared in animals pretreated to deliver antibodies to the basal side of epithelial cells. Once again, antibodies directed against the neurotoxin provided resistance to challenge, but antibodies directed against HA35 did not. Taken collectively, the data indicate that the toxin by itself is capable of crossing epithelial barriers. The data do not support any hypothesis in which HA35 is essential for toxin penetration of epithelial barriers.

Acinetobacter baumannii is a multidrug-resistant (MDR) nosocomial pathogen for which immunotherapeutic alternatives are needed. We previously identified a surface autotransporter of A. baumannii , Ata, that bound to various extracellular matrix/basal membrane proteins and was required for full virulence, biofilm formation, and the adhesion of A. baumannii to collagen type IV. We show here that Ata binding to collagen type IV was inhibited by antibodies to Ata. In addition, in the presence of complement and polymorphonuclear cells (PMNs), antibodies to Ata were highly opsonic against A. baumannii ATCC 17978 and showed low to moderate killing activity against four heterologous A. baumannii strains, whereas in the absence of PMNs, antibody to Ata efficiently promoted complement-dependent bactericidal killing of all of the tested A. baumannii isolates. Using a pneumonia model of infection in both immunocompetent and immunocompromised mice, we found that, compared to normal rabbit sera, antisera to Ata significantly reduced the levels of A. baumannii ATCC 17978 and two MDR strains in the lungs of infected mice. The ability of Ata to engender anti-adhesive, bactericidal, opsonophagocytic, and protective antibodies validates its potential use as an antigenic target against MDR A. baumannii infections.

During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.

The virulence of Bacillus anthraci s, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of the pag , lef , and cya genes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing a pag deletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion of pag or even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism of B. anthracis in the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation of B. anthracis virulence in general and in particular for the design of relevant next-generation anthrax vaccines.

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis . We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon ( capBCADE ), lethal factor ( lef ), edema factor ( cya ), or protective antigen ( pagA ). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA− mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA− mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

The C-terminal 19-kDa domain of merozoite surface protein 1 (MSP1 19 ) is the target of protective antibodies but alone is poorly immunogenic. Previously, using the Plasmodium yoelii murine model, we fused P. yoelii MSP1 19 ( Py MSP1 19 ) with full-length P. yoelii merozoite surface protein 8 (MSP8). Upon immunization, the MSP8-restricted T cell response provided help for the production of high and sustained levels of protective Py MSP1 19 - and Py MSP8-specific antibodies. Here, we assessed the vaccine potential of MSP8 of the human malaria parasite, Plasmodium falciparum . Distinct from Py MSP8, P. falciparum MSP8 ( Pf MSP8) contains an N-terminal asparagine and aspartic acid (Asn/Asp)-rich domain whose function is unknown. Comparative analysis of recombinant full-length Pf MSP8 and a truncated version devoid of the Asn/Asp-rich domain, Pf MSP8(ΔAsn/Asp), showed that both proteins were immunogenic for T cells and B cells. All T cell epitopes utilized mapped within r Pf MSP8(ΔAsn/Asp). The dominant B cell epitopes were conformational and common to both r Pf MSP8 and r Pf MSP8(ΔAsn/Asp). Analysis of native Pf MSP8 expression revealed that Pf MSP8 is present intracellularly in late schizonts and merozoites. Following invasion, Pf MSP8 is found distributed on the surface of ring- and trophozoite-stage parasites. Consistent with a low and/or transient expression of Pf MSP8 on the surface of merozoites, Pf MSP8-specific rabbit IgG did not inhibit the in vitro growth of P. falciparum blood-stage parasites. These studies suggest that the further development of Pf MSP8 as a malaria vaccine component should focus on the use of Pf MSP8(ΔAsn/Asp) and its conserved, immunogenic T cell epitopes as a fusion partner for protective domains of poor immunogens, including Pf MSP1 19 .

The capsule of Neisseria meningitidis is the major virulence factor that enables this bacterium to overcome host immunity elicited by complement and phagocytes, rendering it capable of surviving in blood. As such, nonencapsulated N. meningitidis isolates are generally considered nonpathogenic. Here, we consider the inherent virulence of two nonencapsulated N. meningitidis isolates obtained from our national surveillance of infected blood cultures in Canada. Capsule deficiency of both strains was confirmed by serology and PCR for the ctrA to ctrD genes and siaA to siaC genes, as well as siaD genes specific to serogroups B, C, Y, and W135. In both strains, the capsule synthesis genes were replaced by the capsule null locus, cnl-2 . In accordance with a lack of capsule, both strains were fully susceptible to killing by both human and baby rabbit complement. However, in the presence of cytidine-5′ monophospho- N -acetylneuraminic acid (CMP-NANA), allowing for lipooligosaccharide (LOS) sialylation, a significant increase of resistance to complement killing was observed. Mass spectrometry of purified LOS did not reveal any uncommon modifications that would explain their invasive phenotype. Finally, in a mouse intraperitoneal challenge model, these nonencapsulated isolates displayed enhanced virulence relative to an isogenic mutant of serogroup B strain MC58 lacking capsule (MC58Δ siaD ). Virulence of all nonencapsulated isolates tested was below that of encapsulated serogroup B strains MC58 and B16B6. However, whereas no mortality was observed with MC58Δ siaD , 5/10 mice succumbed to infection with strain 2275 and 2/11 mice succumbed to strain 2274. Our results suggest the acquisition of a new virulence phenotype by these nonencapsulated strains.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli . The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides—CdtA, CdtB, and CdtC—encoded by the genes cdtA , cdtB , and cdtC , respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206–215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA , cdtB , and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens . Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii . Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G 2 /M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.