Louse- and flea-borne rickettsioses: biological and genomic analyses

In contrast to 15 or more validated and/or proposed tick-borne spotted fever group species, only three named medically important rickettsial species are associated with insects. These insect-borne rickettsiae are comprised of two highly pathogenic species, Rickettsia prowazekii (the agent of epidemic typhus) and R. typhi (the agent of murine typhus), as well as R. felis, a species with unconfirmed pathogenicity. Rickettsial association with obligate hematophagous insects such as the human body louse (R. prowazekii transmitted by Pediculus h. humanus) and several flea species (R. typhi and R. felis, as well as R. prowazekii in sylvatic form) provides rickettsiae the potential for further multiplications, longer transmission cycles and rapid spread among susceptible human populations. Both human body lice and fleas are intermittent feeders capable of multiple blood meals per generation, facilitating the efficient transmission of rickettsiae to several disparate hosts within urban/rural ecosystems. While taking into consideration the existing knowledge of rickettsial biology and genomic attributes, we have analyzed and summarized the interacting features that are unique to both the rickettsiae and their vector fleas and lice. Furthermore, factors that underlie rickettsial changing ecology, where native mammalian populations are involved in the maintenance of rickettsial cycle and transmission, are discussed

Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi

The frequency and numbers of Plasmodium falciparum sporozoites transmitted in vitro and corresponding sporozoite loads were determined for experimentally infected Anopheles gambiae and An. stephensi. Geometric mean (GM) sporozoite loads in three experiments ranged from 808 to 13,905 for An. gambiae and from 6,608 to 17,702 for An. stephensi. A total of 44.1% of 68 infected An. gambiae and 49.2% of 63 infected An. stephensi transmitted sporozoites in vitro. The GM number of sporozoites transmitted was 4.5 for An. gambiae and 5.4 for An. stephensi. Overall, 86.9% of the mosquitoes transmitted from one to 25 sporozoites, and only 6.6% transmitted over 100 sporozoites (maximum = 369). Sporozoite loads were not a useful predictor of potential sporozoite transmission. Despite higher sporozoite loads, the numbers of sporozoites transmitted in vitro by the experimentally infected mosquitoes were similar to estimates obtained, using the same techniques, for naturally infected An. gambiae in western Kenya. The low but highly variable numbers of sporozoites transmitted in vitro by mosquitoes used in malaria vaccine challenge studies appears to be a reasonable simulation of natural sporozoite transmission.Peer reviewedEntomology and Plant Patholog

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycocon- jugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycero- phospholipid pathways also initiate from host precursors, and import of both iso- prenes and terpenoids is required for the synthesis of ubiquinone and the lipid car- rier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accen- tuating their parasitic nature. Six biosynthesis pathways contain holes (missing en- zymes); similar patterns in taxonomically diverse bacteria suggest alternative en- zymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host met- abolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell

Geographic Association of Rickettsia felis-Infected Opossums with Human Murine Typhus, Texas

Application of molecular diagnostic technology in the past 10 years has resulted in the discovery of several new species of pathogenic rickettsiae, including Rickettsia felis. As more sequence information for rickettsial genes has become available, the data have been used to reclassify rickettsial species and to develop new diagnostic tools for analysis of mixed rickettsial pathogens. R. felis has been associated with opossums and their fleas in Texas and California. Because R. felis can cause human illness, we investigated the distribution dynamics in the murine typhus–endemic areas of these two states. The geographic distribution of R. felis-infected opossum populations in two well-established endemic foci overlaps with that of the reported human cases of murine typhus. Descriptive epidemiologic analysis of 1998 human cases in Corpus Christi, Texas, identified disease patterns consistent with studies done in the 1980s. A close geographic association of seropositive opossums (22% R. felis; 8% R. typhi) with human murine typhus cases was also observed

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree

The lspA Gene, Encoding the Type II Signal Peptidase of Rickettsia typhi: Transcriptional and Functional Analysis

Lipoprotein processing by the type II signal peptidase (SPase II) is known to be critical for intracellular growth and virulence for many bacteria, but its role in rickettsiae is unknown. Here, we describe the analysis of lspA, encoding a putative SPase II, an essential component of lipoprotein processing in gram-negative bacteria, from Rickettsia typhi. Alignment of deduced amino acid sequences shows the presence of highly conserved residues and domains that are essential for SPase II activity in lipoprotein processing. The transcription of lspA, lgt (encoding prolipoprotein transferase), and lepB (encoding type I signal peptidase), monitored by real-time quantitative reverse transcription-PCR, reveals a differential expression pattern during various stages of rickettsial intracellular growth. The higher transcriptional level of all three genes at the preinfection time point indicates that only live and metabolically active rickettsiae are capable of infection and inducing host cell phagocytosis. lspA and lgt, which are involved in lipoprotein processing, show similar levels of expression. However, lepB, which is involved in nonlipoprotein secretion, shows a higher level of expression, suggesting that LepB is the major signal peptidase for protein secretion and supporting our in silico prediction that out of 89 secretory proteins, only 14 are lipoproteins. Overexpression of R. typhi lspA in Escherichia coli confers increased globomycin resistance, indicating its function as SPase II. In genetic complementation, recombinant lspA from R. typhi significantly restores the growth of temperature-sensitive E. coli Y815 at the nonpermissive temperature, supporting its biological activity as SPase II in prolipoprotein processing