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Species of genus Rickettsia are obligate intracellular bacterial parasites of a wide range of arthropod and vertebrate hosts. Some Rickettsia species are responsible for several serious human diseases. One fascinating feature of these stealthy group of pathogens is their ability to exploit host cytosolic defense responses to their benefits. However, the precise mechanism by which pathogenic Rickettsia elude host immune defense responses remains to be determined. Here, we observed that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis become ubiquitinated and induce autophagy upon entry into bone-marrow-derived macrophages. Moreover, unlike R. montanensis, R. typhi, and R. rickettsii colocalized with LC3B but not with Lamp2 upon host cell entry. Finally, we observed that pathogenic but not non-pathogenic Rickettsia reduce pro-inflammatory IL-1 responses. In sum, we identified a previously unappreciated pathway by which pathogenic, but not non-pathogenic, Rickettsia become ubiquitinated and induced autophagy, but avoided autophagolysosomal destruction as well as inflammasome-mediated anti-microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages.

Rickettsia species are diverse Gram-negative obligate intracellular bacteria often pervasive in numerous invertebrates, as well as fungal, nematode and microeukaryotic hosts. Certain species are etiological agents for well-known arthropod-borne illnesses; e.g., R. rickettsii (Rocky Mountain Spotted Fever), R. prowazekii (Epidemic Typhus), and R. typhi, (Endemic Typhus). Living freely in eukaryotic cytosol presumably exposes rickettsiae to host cell immune receptors, particularly those recognizing bacterial cell envelope glycoconjugates. However, the mechanics of host recognition of rickettsiae remain poorly defined. As rickettsiae synthesize a canonical Gram-negative cell envelope that includes peptidoglycan (PGN) and lipopolysaccharide (LPS), structural insight on these macromolecules is important for deciphering host responses to these pathogens. In this work, PGN from R. typhi was digested and the resultant subunits were analyzed by two different, albeit complementary, sample preparation methods. Both approaches were subsequently subjected to liquid chromatography/mass spectrometry analysis to infer PGN structure. R. typhi PGN was determined to be similar to most other Gram-negative bacteria, with mDAP-type muropeptide subunits. However, additional alanine residues were observed elongating the muropeptide stems, rather than the glycine residues usually observed in Gram-negative bacterial PGN. Despite this deviation, R. typhi contains a murein layer that is predicted to agonize host cellular PGN receptors and be susceptible to PGN-targeting antimicrobials. This same structure is likely synthesized by all Rickettsia species, as bioinformatics and comparative genomics analyses indicate the biosynthesis of PGN is highly conserved. Determining how host cells process this canonical glycoconjugate during infection is crucial for identifying factors behind rickettsial pathogenesis, including immunoavoidance or proinflammatory mechanisms possibly employed by rickettsiae with varying pathogenic potential.

Rickettsiaspecies (spp.) are strict obligate intracellular bacteria, with some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding ofRickettsiacell biology and pathogenesis are evolving, the mechanisms by which pathogenicRickettsiaspp. evade host innate immune detection remains elusive. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected withR. typhi(etiologic agent of murinetyphus) andR. rickettsii(etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenicR. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1α and to a lesser extent IL- 1β. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infectedWTmice. As macrophages are a primary source of both IL-1α and IL-1β cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenicR. typhiandR. rickettsii, but not non-pathogenicR. montanensis, eluded pro- IL-1α induction and benefited pre-dominantly from the reduced IL-1α secretion, via a Caspase-11-Gsdmd-dependent pathway, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis inWTmice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1α signaling axis in macrophages thus supporting their replication within the host.IMPORTANCECurrently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenicRickettsiaspecies circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1α signaling axis, to limiting the replication of pathogenicR. rickettsiaandR. typhispecies in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptative transfer studies further identified IL-1α-secreting macrophages as critical mediators in controlling rickettsial infection inWTmice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenicRickettsiaspp., benefit from a reduction in the Caspase-11-Gsdmd-mediated release of IL-1α to support host colonization.

Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive., Species of Rickettsia (Alphaproteobacteria: Rickettsiales) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses. IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies.

Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host., Rickettsiae deploy their effector arsenal to manipulate membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms; to gain access, and promote their intracytosolic lifespan to ultimately expedite transmission.

Additional file 1: Figure S1. Assessing assembly fragmentation, gene duplication and repeat elements within the C. felis assembly. (A) Evaluating assembly fragmentation via mapping of scaffolds shorter than 1 Mb (n = 3724) to scaffolds larger than 1 Mb (n = 9, “BIG9 scaffolds”). All but 2 short scaffolds mapped to a BIG9 scaffold at least once; confidence intervals are based on the probability of mapping to a single unique location. (B). Assessing the “genome completeness” of the C. felis full assembly and BIG9 scaffolds through comparison to eukaryote, arthropod and insect BUSCOs. (C) Tandem and proximal duplicate gene locations on BIG9 scaffold 1, (D) BIG9 scaffold 2, (E) BIG9 scaffold 3, (F) BIG9 scaffold 4, (G) BIG9 scaffold 5, (H) BIG9 scaffold 6, (I) BIG9 scaffold 7, (J) BIG9 scaffold 8, (K) BIG9 scaffold 9. (L) Duplications by proximity. Only true duplications (n = 2) are shown. Red bars (*) depict “dispersed” clusters that span multiple scaffolds. (M) Dispersed duplicate gene locations across BIG9 scaffolds. (N) Distribution across BIG9 scaffolds of C. felis proteins annotated as “DNA integration” (GO:0015074, see Additional file 2: Table S1. for specific accession numbers) and their relation to gene duplications. (O) Compilation of retroelements, DNA transposons and other repeat elements predicted across the BIG9 scaffolds. Overall totals are highlighted yellow. (P) tRNA gene abundances and (Q) codon usage/amino acid for select Holometabola.

Additional file 3: Figure S2. Representative histograms produced by flow cytometry showing the peak positions of the 2C nuclei of Drosophila melanogaster (left) and D. virilis (center) female standards, and individual C. felis females (right) from the sequenced EL strain. (A) A 434 Mb flea. (B) A 553 Mb flea. All peaks have CV 500 nuclei under the statistical gates (red lines spanning the 2C peaks).

Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P 2 , by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P 2 for R. typhi entry. PI(3,4,5)P 3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P 3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism.

Background Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. Results By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433–551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable “genome-in-flux” phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. Conclusion Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts—potential tools for blocking pathogen transmission—these oddities highlight a unique and underappreciated disease vector.

Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 in R. rickettsii Iowa converts this avirulent strain’s typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence., IMPORTANCE Members of the genus Rickettsia are obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain of R. rickettsii, the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.

Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized in Rickettsia pathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in both Rickettsia typhi and Rickettsia rickettsii, the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C14/C16) to the 3′-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis. IMPORTANCE Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, from Rickettsia typhi and R. rickettsii catalyzes the addition of C16 fatty acid chains into the lipid A 3′-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A of Escherichia coli.

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component of Rickettsia biology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borne Rickettsia typhi , the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regarding R. typhi biology in flea vectors that, importantly, do not suffer lethality due to R. typhi infection. To address if fleas combat rickettsial infection, we characterized the cat flea ( Ctenocephalides felis ) innate immune response to R. typhi . Initially, we determined that R. typhi infects Drosophila cells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of the C. felis transcriptome identified homologs to all of the Drosophila immune deficiency (IMD) and Toll pathway components, an AMP gene expression profile in Drosophila cells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessed R. typhi -mediated flea IMD pathway activation in vivo using small interfering RNA (siRNA)-mediated knockdown. Knockdown of Relish and Imd increased R. typhi infection levels, implicating the IMD pathway as a critical regulator of R. typhi burden in C. felis . These data suggest that targeting the IMD pathway could minimize the spread of R. typhi , and potentially other human pathogens, vectored by fleas.

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 glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes 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 metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell., IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia, arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

Many prokaryotes utilize type IV secretion systems (T4SSs) to translocate substrates (e.g. nucleoprotein, DNA, protein) across the cell envelope, and/or to elaborate surface structures (i.e. pili or adhesins). Among eight distinct T4SS classes, P-T4SSs are typified by the Agrobacterium tumefaciens vir T4SS, which is comprised of 12 scaffold components (VirB1-VirB11, VirD4). While most P-T4SSs include all 12 Vir proteins, some differ from the vir archetype by either containing additional scaffold components not analogous to Vir proteins or lacking one or more of the Vir proteins. In a special case, the Rickettsiales vir homolog (rvh) P-T4SS comprises unprecedented gene family expansion. rvh contains three families of gene duplications (rvhB9, rvhB8, rvhB4): RvhB9,8,4-I are conserved relative to equivalents in other P-T4SSs, while RvhB9,8,4-II have evolved atypical features that deviate substantially from other homologs. Furthermore, rvh contains five VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Our work herein focuses on the complexity underpinned by rvh gene family expansion. Furthermore, we describe an RvhB10 insertion, which occurs in a region that forms the T4SS pore. The significance of these curious properties to rvh structure and function is evaluated, shedding light on a highly complex T4SS.

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium., IMPORTANCE Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.

A defining facet of tick- Rickettsia symbioses is the molecular strategy employed by each partner to ensure its own survival. Ticks must control rickettsial colonization to avoid immediate death. In the current study, we show that rickettsial abundance in the tick midgut increases once the expression of a Kunitz-type serine protease inhibitor from the American dog tick ( Dermacentor variabilis ) (DvKPI) is suppressed by small interfering RNA (siRNA). A series of in vitro invasion assays suggested that DvKPI limits rickettsial colonization during host cell entry. Interestingly, we observed that DvKPI associates with rickettsiae in vitro as well as in the tick midgut. Collectively, our data demonstrate that DvKPI limits host cell invasion by Rickettsia montanensis , possibly through an association with the bacterium.

Phospholipase A 2 (PLA 2 ) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA 2 activity has not been identified or functionally characterized. Here, we report that the Rickettsia typhi genome possesses two genes encoding patatin-like PLA 2 proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA 2 activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of R. typhi in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during R. typhi infection of host cells. In silico , RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA 2 inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from Rickettsia typhi with functional phospholipase A activity.

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases., Author Summary Phylogenomics analysis indicates divergent mechanisms for host cell invasion across diverse species of obligate intracellular Rickettsia. For instance, only some Rickettsia species carry RalF, the rare bacterial Arf-GEF effector utilized by Legionella pneumophila to facilitate fusion of ER-derived membranes with its host-derived vacuole. For R. prowazekii (Typhus Group, TG), prior in vitro studies suggested the Arf-GEF activity of RalF, which is absent from Spotted Fever Group species, might be spatially regulated at the host plasma membrane. Herein, we demonstrate RalF of R. typhi (TG) and R. felis (Transitional Group) localizes to the host plasma membrane, yet R. bellii (Ancestral Group) RalF shows perinuclear localization reminiscent of RalF-mediated recruitment of Arf1 by L. pneumophila to its vacuole. For R. typhi, RalF expression occurs early during infection, with RalF inactivation significantly reducing host cell invasion. Furthermore, RalF co-localization with Arf6 and the phosphoinositide PI(4,5)P2 at the host plasma membrane was determined to be critical for R. typhi invasion. Thus, our work illustrates that different intracellular lifestyles across species of Rickettsia and Legionella have driven divergent roles for RalF during host cell infection. Collectively, we identify lineage-specific Arf-GEF utilization across diverse rickettsial species, previously unappreciated mechanisms for host cell invasion and infection.

While examining the molecular basis for the lack of actin-based motility for the non-pathogenic spotted fever group (SFG) R. peacockii, we identified a novel insertion sequence (IS) element, ISRpe1, disrupting the coding sequence of rickA, demonstrated to induce actin-tail polymerization for the SFG rickettsiae. This rickettsial IS element appears to be active in that complete terminal inverted repeat and recombinase/transposase open reading frame sequences are present and the transposase is transcriptionally expressed. Phylogenetically, ISRpe1 belongs to a new IS family that is most closely related to those transposable elements of other intracellular bacteria like Wolbachia spp. ISRpe1 was demonstrated to be present in at least 10 locations throughout the R. peacockii genome, including one that disrupted the putative cell surface antigen encoding gene, sca1 considered to be involved in adhesion and virulence of the rickettsiae. Additionally, three IS sites demonstrated rearrangements/relocations of the R. peacockii genome when compared to those of other SFG rickettsiae. Our findings of the disruptions of rickA and sca1 along with the comparative genomic reassortments associated with ISRpe1 in the non-virulent R. peacockii provides opportunities to uncover molecular mechanisms underlying the pathogenesis and evolution of rickettsiae as well as its potential to be used in rickettsial transposon-based mutagenesis.

The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4-ethenocytosine (eC) lesions borne on transfected single-stranded M13 vector DNA. All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at eC residues. In contrast to normal cells that show ≈ 6% mutagenesis at eC lesions, but ≈ 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at eC lesion sites in untreated cells. However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that eC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at eC can also be independently elevated by the SOS pathway.

The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’.

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N 4 -ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac -derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species., Author Summary Typhus Group (TG) rickettsiae include Rickettsia typhi and R. prowazekii, the etiological agents of murine and epidemic typhus, respectively. The rickettsial obligate intracellular life cycle complicates conventional approaches to genetic manipulation, impeding characterization of molecules underlying pathogenesis. Phospholipase A2 (PLA2) enzymes are factors that likely mediate such processes as host entry, phagosomal escape, and host lysis for cell-to-cell spread. Here, we demonstrate across 46 Rickettsia genomes that genes encoding two PLA2 enzymes (pat1 and pat2) have divergent evolutionary histories and discordant selective constraints, with pat2 deleted from most other non-TG rickettsiae genomes. As previously determined for Pat2, we show here that R. typhi Pat1 is secreted into the host cell cytoplasm during infection and requires a host cofactor for enzymatic activity. Like Pat2, recombinant Pat1 protein is cytotoxic to yeast cells. Pretreatment of R. typhi with anti-Pat1 or anti-Pat2 antibody results in a significant decrease in rickettsial infection, implicating roles for both Pat1 and Pat2 during the early stage of R. typhi host cell infection. Collectively, our work suggests that R. typhi utilizes two evolutionary divergent phospholipases during its intracellular life cycle, a mechanism distinguishing TG rickettsiae-associated cell biology and pathogenesis from other rickettsioses, particularly those associated with Spotted Fever Group pathogens.

Rickettsia typhi , the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene ( RT0218 ) encoding a C-terminal ankyrin repeat domain-containing protein, named R ickettsia a nkyrin r epeat p rotein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi . RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli . Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1.

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi , the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli -based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli , and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

Recent studies aimed at elucidating the rickettsia-tick interaction have discovered that the spotted fever group rickettsia Rickettsia montanensis , a relative of R. rickettsii , the etiologic agent of Rocky Mountain spotted fever, induces differential gene expression patterns in the ovaries of the hard tick Dermacentor variabilis . Here we describe a new defensin isoform, defensin-2, and the expression patterns of genes for three antimicrobials, defensin-1 ( vsnA1 ), defensin-2, and lysozyme, in the midguts and fat bodies of D. variabilis ticks that were challenged with R. montanensis . Bioinformatic and phylogenetic analyses of the primary structure of defensin-2 support its role as an antimicrobial. The tissue distributions of the three antimicrobials, especially the two D. variabilis defensin isoforms, are markedly different, illustrating the immunocompetence of the many tissues that R. montanensis presumably invades once acquired by the tick. Antimicrobial gene expression patterns in R. montanensis -challenged ticks suggest that antimicrobial genes play a role during the acquisition-invasion stages in the tick.

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

While examining the molecular basis for the lack of actin-based motility for the non-pathogenic spotted fever group (SFG) R. peacockii, we identified a novel insertion sequence (IS) element, ISRpe1, disrupting the coding sequence of rickA, demonstrated to induce actin-tail polymerization for the SFG rickettsiae. This rickettsial IS element appears to be active in that complete terminal inverted repeat and recombinase/transposase open reading frame sequences are present and the transposase is transcriptionally expressed. Phylogenetically, ISRpe1 belongs to a new IS family that is most closely related to those transposable elements of other intracellular bacteria like Wolbachia spp. ISRpe1 was demonstrated to be present in at least 10 locations throughout the R. peacockii genome, including one that disrupted the putative cell surface antigen encoding gene, sca1 considered to be involved in adhesion and virulence of the rickettsiae. Additionally, three IS sites demonstrated rearrangements/relocations of the R. peacockii genome when compared to those of other SFG rickettsiae. Our findings of the disruptions of rickA and sca1 along with the comparative genomic reassortments associated with ISRpe1 in the non-virulent R. peacockii provides opportunities to uncover molecular mechanisms underlying the pathogenesis and evolution of rickettsiae as well as its potential to be used in rickettsial transposon-based mutagenesis.

The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 °C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 °C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.

The type I signal peptidaselepBgenes fromRickettsia rickettsiiandRickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the clonedlepBgenes fromR. rickettsiiandR. typhishows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that thelepBgene ofR. rickettsiiis cotranscribed in a polycistronic message with the putativenuoF(encoding NADH dehydrogenase I chain F),secF(encoding protein export membrane protein), andrnc(encoding RNase III) genes in asecF-nuoF-lepB-rnccluster. The clonedlepBgenes fromR. rickettsiiandR. typhihave been demonstrated to possess signal peptidase I activity inEscherichia colipreprotein processing in vivo by complementation assay.

The groESL operon from an obligate, intracellular, Gram-negative bacterium Rickettsia typhi, the etiologic agent of murine typhus, was cloned and sequenced. The sequence analysis of 2229 bp of the groESL operon reveals two open reading frames of 288 nucleotides (groES) and 1653 nucleotides (groEL) separated by 20 nucleotides. The deduced amino acid sequence of R. typhi GroES and GroEL shows a high degree of identity with other bacterial GroES and GroEL. Reverse transcriptase-polymerase chain reaction and Northern blot analysis indicated that both groES and groEL are transcribed as a single mRNA. The transcriptional start point at 81 nucleotides upstream of the groES start codon was determined by primer extension. The promoter analysis shows no regulatory CIRCE element as it is known for many Gram-positive and Gram-negative bacteria. However, it contains the sequence similar to the putative sigma(70)-dependent promoter and lacks the -35 sequence of the putative sigma(32)-dependent promoter. Complementation assay by R. typhi groESL in a temperature sensitive Escherichia coli groEL mutant restored significant growth ability at non-permissive temperature.

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.

Nebularine (9-β- d -ribofuranosylpurine; Nb) is a naturally occurring nucleoside with structural features suggestive of a universal base. However, previous observations based on thermal melting characteristics of oligonucleotides suggested that Nb formed stable pairs only with thymine. To determine the template characteristics of Nb, we constructed M13 viral single-stranded DNA (ssDNA) molecules bearing a single site-specific deoxynebularine (9-2′-deoxy-β- d -ribofuranosylpurine) residue. The ssDNA constructs were transfected into Escherichia coli cells to determine the specificity of base insertion opposite Nb, as well as to determine the effect of Nb on the replicability of the transfected DNA. Base insertion opposite Nb, analyzed by a multiplex sequencing technology, suggests that Nb has the template characteristics of adenine. Analysis of DNA replicability, measured as transfection efficiency, indicates that Nb does not block DNA replication. UV irradiation of host cells before transfection did not significantly affect survival or base insertion specificity within the limits of multiplex sequencing technology employed, suggesting that inducible mutagenic phenomena appear to have only minor effects on translesion synthesis across Nb. In addition, in vitro DNA elongation experiments on oligonucleotide templates using E. coli DNA polymerase I (Klenow fragment) as the model polymerase showed that Nb templates for T, but not for other bases under the tested conditions. The data reported in this communication underscore the importance of base-pair geometry as a specificity-determinant during base insertion by replicative polymerases.

Mutation fixation at an ethenocytosine (epsilon C) residue borne on transfected M13 single-stranded DNA is significantly enhanced in response to pretreatment of Escherichia coli cells with UV, alkylating agents or hydrogen peroxide, a phenomenon that we have called UVM for UV modulation of mutagenesis. The UVM response does not require the E. coli SOS or adaptive responses, and is observed in cells defective for oxyR, an oxidative DNA damage-responsive regulatory gene. UVM may represent either a novel DNA-repair phenomenon, or an unrecognized feature of DNA replication in damaged cells that affects a specific class of non-coding DNA lesions. To explore the range of DNA lesions subject to the UVM effect, we have examined mutation fixation at 3,N4-ethenocytosine and 1,N6-ethenoadenine, as well as at O6-methylguanine (O6mG). M13 viral single-stranded DNA constructs bearing a single mutagenic lesion at a specific site were transfected into cells pretreated with UV or 1-methyl-3-nitro-1-nitroso-guanidine (MNNG). Survival of transfected viral DNA was measured as transfection efficiency, and mutagenesis at the lesion site was analysed by a quantitative multiplex sequence analysis technology. The results suggest that the UVM effect modulates mutagenesis at the two etheno lesions, but does not appear to significantly affect mutagenesis at O6mG. Because the modulation of mutagenesis is observed in cells incapable of the SOS response, these data are consistent with the notion that UVM may represent a previously unrecognized DNA damage-inducible response that affects the fidelity of DNA replication at certain mutagenic lesions in Escherichia coli.

Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1–3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host., Author Summary Rickettsia typhi, a member of the typhus group (TG) rickettsia, is the agent of murine or endemic typhus fever – a disease exhibiting mild to severe flu-like symptoms resulting in significant morbidity. It is maintained in a flearodent transmission cycle in urban and suburban environments. The obligate intracellular lifestyle of rickettsiae makes genetic manipulation difficult and impedes progress towards identification of virulence factors. All five Scas were detected on the surface of R.. typhi using a combination of a biotin-labeled affinity assay, negative stain electron microscopy and flow cytometry. Sca proteins are members of the autotransporter (AT) family or type V secretion system (TVSS). We employed detailed bioinformatic analyses and evaluated their transcript abundance in an in vitro infection model where sca transcripts are detected at varying levels over the course of a 5 day in vitro infection. We also observe expression of selected Sca proteins during infection of fleas and rats. Our study provides a proteomic analysis of the bacterial surface and an initial characterization of the Sca family as it exists in R. typhi.