Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis.

Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

Hereditary hemochromatosis restores the virulence of plague vaccine strains.

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.

Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines.

Yersinia pestis causes plague, a disease with high mortality in humans that can be transmitted by fleabite or aerosol. A US Food and Drug Administration (FDA)-licensed plague vaccine is currently not available. Vaccine developers have focused on two subunits of Y. pestis: LcrV, a protein at the tip of type III secretion needles, and F1, the fraction 1 pilus antigen. F1-V, a hybrid generated via translational fusion of both antigens, is being developed for licensure as a plague vaccine. The rV10 vaccine is a non-toxigenic variant of LcrV lacking residues 271-300. Here we developed Current Good Manufacturing Practice (cGMP) protocols for rV10. Comparison of clinical grade rV10 with F1-V did not reveal significant differences in plague protection in mice, guinea pigs or cynomolgus macaques. We also developed cGMP protocols for rV10-2, a variant of rV10 with an altered affinity tag. Immunization with rV10-2 adsorbed to aluminum hydroxide elicited antibodies against LcrV and conferred pneumonic plague protection in mice, rats, guinea pigs, cynomolgus macaques and African Green monkeys. The data support further development of rV10-2 for FDA Investigational New Drug (IND) authorization review and clinical testing.

The Rickettsia conorii autotransporter protein Sca1 promotes adherence to nonphagocytic mammalian cells.

The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells.

Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner.

Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.