Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-ß signaling in primary human dermal microvascular endothelial cells.

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-ß secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-ß secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-ß production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-ß production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-ß response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-ß secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-ß response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution & Composition in Bacterial Aggregates.

Bacteria in natural environments and infections are often found in cell aggregates suspended in polymer-rich solutions, and aggregation can promote bacterial survival and stress resistance. One aggregation mechanism, called depletion aggregation, is driven by physical forces between bacteria and high concentrations of polymers in the environment rather than bacterial activity per se. As such, bacteria aggregated by the depletion mechanism will disperse when polymer concentrations fall unless other adhesion mechanisms supervene. Here we investigated whether the depletion mechanism can actuate the aggregating effects of Pseudomonas aeruginosa exopolysaccharides for suspended (i.e. not surface attached) bacteria, and how depletion affects bacterial inter-species interactions. We found that cells overexpressing the exopolysaccharides Pel and Psl remained aggregated after short periods of depletion aggregation whereas wild-type and mucoid P. aeruginosa did not. In co-culture, depletion aggregation had contrasting effects on P. aeruginosa's interactions with coccus- and rod-shaped bacteria. Depletion caused S. aureus (cocci) and P. aeruginosa (rods) to segregate from each other and S. aureus to resist secreted P. aeruginosa antimicrobial factors resulting in species co-existence. In contrast, depletion aggregation caused P. aeruginosa and Burkholderia sp. (both rods) to intermix, enhancing type VI secretion inhibition of Burkholderia by P. aeruginosa, leading to P. aeruginosa dominance. These results show that in addition to being a primary cause of aggregation in polymer-rich suspensions, physical forces inherent to the depletion mechanism can promote aggregation by some self-produced exopolysaccharides and determine species distribution and composition of bacterial communities.

Evolutionary relationships among Massospora spp. (Entomophthorales), obligate pathogens of cicadas.

The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree (EFL) or a basal polytomy (28S, BTUB). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Measures to Control Phlebotomus argentipes and Visceral Leishmaniasis in India.

Visceral leishmaniasis is a deadly parasitic disease that is transmitted via the bite of a female sand fly, Phlebotomus argentipes. The highest burden of this disease is in northern India. In 2005, India embarked on an initiative with Nepal, Bangladesh, and the World Health Organization to eliminate visceral leishmaniasis by 2015. With the goal of 1 case in 10,000 people still unmet, it is prudent to evaluate the tools that have been used thus far to reduce vector numbers and cases of the disease. Herein, we present a review of studies conducted on vector-control strategies in India to combat visceral leishmaniasis including indoor residual spraying, insecticide-treated bed nets, environmental modification, and feed-through insecticides. This review suggests that the quality of indoor residual spraying may enhance control measures while a combination of spraying, nets, and feed-through insecticides would best confront the diverse habitats of P. argentipes.

Comparative genome sequencing of Rickettsia rickettsii strains that differ in virulence.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii, the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

Pathogenic enterobacteria in lemurs associated with anthropogenic disturbance.

As human population density continues to increase exponentially, speeding the reduction and fragmentation of primate habitat, greater human-primate contact is inevitable, making higher rates of pathogen transmission likely. Anthropogenic effects are particularly evident in Madagascar, where a diversity of endemic lemur species are threatened by rapid habitat loss. Despite these risks, knowledge of how anthropogenic activities affect lemur exposure to pathogens is limited. To improve our understanding of this interplay, we non-invasively examined six species of wild lemurs in Ranomafana National Park for enteric bacterial pathogens commonly associated with diarrheal disease in human populations in Madagascar. Patterns of infection with Enterotoxigenic Escherichia coli, Shigella spp., Salmonella enterica, Vibrio cholerae, and Yersinia spp. (enterocolitica and pseudotuberculosis) were compared between lemurs inhabiting intact forest and lemurs inhabiting degraded habitat with frequent exposure to tourism and other human activity. Fecal samples acquired from humans, livestock, and rodents living near the degraded habitat were also screened for these bacteria. Remarkably, only lemurs living in disturbed areas of the park tested positive for these pathogens. Moreover, all of these pathogens were present in the human, livestock, and/or rodent populations. These data suggest that lemurs residing in forests altered or frequented by people, livestock, or peridomestic rodents, are at risk for infection by these diarrhea-causing enterobacteria and other similarly transmitted pathogens.

Epidemiology of pathogenic enterobacteria in humans, livestock, and peridomestic rodents in rural Madagascar.

BACKGROUND: Among the families of enteric bacteria are globally important diarrheal agents. Despite their potential for zoonotic and environmental transmission, few studies have examined the epidemiology of these pathogens in rural systems characterized by extensive overlap among humans, domesticated and peridomestic animals. We investigated patterns of infection with Enterotoxigenic Escherichia coli, Shigella spp., Salmonella enterica, Vibrio cholerae, and Yersinia spp. (enterocolitica, and pseudotuberculosis) in Southeastern Madagascar where the potential for the aforementioned interactions is high. In this pilot project we conducted surveys to examine behaviors potentially associated with risk of infection and if infection with specific enterobacteria species was associated with diarrheal disease. METHODOLOGY/PRINCIPAL FINDINGS: PCR was conducted on DNA from human, livestock, and rodent fecal samples from three villages. Overall, human prevalence was highest (77%), followed by rodents (51%) and livestock (18%). Rodents were ∼2.8 times more likely than livestock to carry one of the bacteria. The incidence of individual species varied between villages, with the observation that, E. coli and Shigella spp. were consistently associated with co-infections. As an aggregate, there was a significant risk of infection linked to a water source in one village. Individually, different pathogens were associated with certain behaviors, including: those who had used medication, experienced diarrhea in the past four weeks, or do not use toilets. CONCLUSIONS/SIGNIFICANCE: Different bacteria were associated with an elevated risk of infection for various human activities or characteristics. Certain bacteria may also predispose people to co-infections. These data suggest that a high potential for transmission among these groups, either directly or via contaminated water sources. As these bacteria were most prevalent in humans, it is possible that they are maintained in humans and that transmission to other species is infrequent. Further studies are needed to understand bacterial persistence, transmission dynamics, and associated consequences in this and similar systems.

Francisella tularensis suppresses the proinflammatory response of endothelial cells via the endothelial protein C receptor.

Various bacterial pathogens activate the endothelium to secrete proinflammatory cytokines and recruit circulating leukocytes. In contrast, there is a distinct lack of activation of these cells by Francisella tularensis, the causative agent of tularemia. Given the importance of endothelial cells in facilitating innate immunity, we investigated the ability of the attenuated live vaccine strain and virulent Schu S4 strain of F. tularensis to inhibit the proinflammatory response of HUVECs. Living F. tularensis live vaccine strain and Schu S4 did not stimulate secretion of the chemokine CCL2 by HUVECs, whereas material released from heat-killed bacteria did. Furthermore, the living bacteria suppressed secretion in response to heat-killed F. tularensis. This phenomenon was dose and contact dependent, and it occurred rapidly upon infection. The living bacteria did not inhibit the activation of HUVECs by Escherichia coli LPS, highlighting the specificity of this suppression. The endothelial protein C receptor (EPCR) confers anti-inflammatory properties when bound by activated protein C. When the EPCR was blocked, F. tularensis lost the ability to suppress activation of HUVECs. To our knowledge, this is the first report that a bacterial pathogen inhibits the host immune response via the EPCR. Endothelial cells are a critical component of the innate immune response to infection, and suppression of their activation by F. tularensis is likely a mechanism that aids in bacterial dissemination and evasion of host defenses.

GroEL and lipopolysaccharide from Francisella tularensis live vaccine strain synergistically activate human macrophages.

Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 microg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 microg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 microg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium.

A tolC mutant of Francisella tularensis is hypercytotoxic compared to the wild type and elicits increased proinflammatory responses from host cells.

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS DeltatolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the DeltatolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The DeltatolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.