Evolution of an Iron-Detoxifying Protein: Eukaryotic and Rickettsia Frataxins Contain a Conserved Site Which Is Not Present in Their Bacterial Homologues.

Friedreich's ataxia is a neurodegenerative disease caused by mutations in the frataxin gene. Frataxin homologues, including bacterial CyaY proteins, can be found in most species and play a fundamental role in mitochondrial iron homeostasis, either promoting iron assembly into metaloproteins or contributing to iron detoxification. While several lines of evidence suggest that eukaryotic frataxins are more effective than bacterial ones in iron detoxification, the residues involved in this gain of function are unknown. In this work, we analyze conservation of amino acid sequence and protein structure among frataxins and CyaY proteins to identify four highly conserved residue clusters and group them into potential functional clusters. Clusters 1, 2, and 4 are present in eukaryotic frataxins and bacterial CyaY proteins. Cluster 3, containing two serines, a tyrosine, and a glutamate, is only present in eukaryotic frataxins and on CyaY proteins from the Rickettsia genus. Residues from cluster 3 are blocking a small cavity of about 40 Å present in E. coli's CyaY. The function of this cluster is unknown, but we hypothesize that its tyrosine may contribute to prevent formation of reactive oxygen species during iron detoxification. This cluster provides an example of gain of function during evolution in a protein involved in iron homeostasis, as our results suggests that Cluster 3 was present in the endosymbiont ancestor of mitochondria and was conserved in eukaryotic frataxins.

A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

The Ankyrin Repeat Protein RARP-1 Is a Periplasmic Factor That Supports Rickettsia parkeri Growth and Host Cell Invasion.

Rickettsia spp. are obligate intracellular bacterial pathogens that have evolved a variety of strategies to exploit their host cell niche. However, the bacterial factors that contribute to this intracellular lifestyle are poorly understood. Here, we show that the conserved ankyrin repeat protein RARP-1 supports Rickettsia parkeri infection. Specifically, RARP-1 promotes efficient host cell entry and growth within the host cytoplasm, but it is not necessary for cell-to-cell spread or evasion of host autophagy. We further demonstrate that RARP-1 is not secreted into the host cytoplasm by R. parkeri. Instead, RARP-1 resides in the periplasm, and we identify several binding partners that are predicted to work in concert with RARP-1 during infection. Altogether, our data reveal that RARP-1 plays a critical role in the rickettsial life cycle. IMPORTANCERickettsia spp. are obligate intracellular bacterial pathogens that pose a growing threat to human health. Nevertheless, their strict reliance on a host cell niche has hindered investigation of the molecular mechanisms driving rickettsial infection. This study yields much-needed insight into the Rickettsia ankyrin repeat protein RARP-1, which is conserved across the genus but has not yet been functionally characterized. Earlier work had suggested that RARP-1 is secreted into the host cytoplasm. However, the results from this work demonstrate that R. parkeri RARP-1 resides in the periplasm and is important both for invasion of host cells and for growth in the host cell cytoplasm. These results reveal RARP-1 as a novel regulator of the rickettsial life cycle.

Regulator of Actin-Based Motility (RoaM) Downregulates Actin Tail Formation by Rickettsia rickettsii and Is Negatively Selected in Mammalian Cell Culture.

The etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is an obligately intracellular pathogen that induces the polymerization of actin filaments to propel the bacterium through the cytoplasm and spread to new host cells. Cell-to-cell spread via actin-based motility is considered a key virulence determinant for spotted fever group rickettsiae, as interruption of sca2, the gene directly responsible for actin polymerization, has been shown to reduce fever in guinea pigs. However, little is known about how, or if, motility is regulated by the bacterium itself. We isolated a hyperspreading variant of R. rickettsii Sheila Smith that produces actin tails at an increased rate. A1G_06520 (roaM [regulator of actin-based motility]) was identified as a negative regulator of actin tail formation. Disruption of RoaM significantly increased the number of actin tails compared to the wild-type strain but did not increase virulence in guinea pigs; however, overexpression of RoaM dramatically decreased the presence of actin tails and moderated fever response. Localization experiments suggest that RoaM is not secreted, while reverse transcription-quantitative PCR (RT-qPCR) data show that various levels of RoaM do not significantly affect the expression of the known rickettsial actin-regulating proteins sca2, sca4, and rickA. Taken together, the data suggest a previously unrecognized level of regulation of actin-based motility in spotted fever group rickettsiae. Although this gene is intact in many isolates of spotted fever, transitional, and ancestral group Rickettsia spp., it is often ablated in highly passaged laboratory strains. Serial passage experiments revealed strong negative selection of roaM in Vero 76 cells. IMPORTANCE The mechanism of actin-based motility of spotted fever group Rickettsia has been studied extensively, but here, we provide genetic evidence that motility is a regulated process in R. rickettsii. The findings also suggest that serial passage of rickettsial strains in cell culture may cause the bacteria to lose essential genes that are no longer conserved under natural selective pressure. These findings are likely relevant to the interpretation of studies concerning virulence determinants of rickettsiae.

The enigmatic biology of rickettsiae: recent advances, open questions and outlook.

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses and are among the oldest known vector-borne pathogens. Members of this genus are extraordinarily diverse and exhibit a broad host range. To establish intracellular infection, Rickettsia species undergo complex, multistep life cycles that are encoded by heavily streamlined genomes. As a result of reductive genome evolution, rickettsiae are exquisitely tailored to their host cell environment but cannot survive extracellularly. This host-cell dependence makes for a compelling system to uncover novel host-pathogen biology, but it has also hindered experimental progress. Consequently, the molecular details of rickettsial biology and pathogenesis remain poorly understood. With recent advances in molecular biology and genetics, the field is poised to start unraveling the molecular mechanisms of these host-pathogen interactions. Here, we review recent discoveries that have shed light on key aspects of rickettsial biology. These studies have revealed that rickettsiae subvert host cells using mechanisms that are distinct from other better-studied pathogens, underscoring the great potential of the Rickettsia genus for revealing novel biology. We also highlight several open questions as promising areas for future study and discuss the path toward solving the fundamental mysteries of this neglected and emerging human pathogen.

Rickettsia-host interaction: strategies of intracytosolic host colonization.

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.

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells.

BACKGROUND: Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. RESULTS: We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR's), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5'-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. CONCLUSIONS: In sum, the findings of this study authenticate the presence of novel Rc_sR's in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis.

The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1-6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7-11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15-17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.

A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

Optimized fixation of actin filaments for improved indirect immunofluorescence staining of rickettsiae.

OBJECTIVE: The objective was to investigate fixative solutions: 3.7% formaldehyde, 4% paraformaldehyde, 4% paraformaldehyde in the cytoskeletal buffer and 4% paraformaldehyde in PHEM buffer (containing PIPES, HEPES, EGTA and MgCl2), applicable for immunofluorescence assay. RESULTS: Herein we optimized this serological technique, testing four fixative solutions, for the sensitive detection of rickettsial antigens, and preservation of intracellular structures of the host cells, particularly filamentous actin. Rickettsial antigens were presented equally well both with formaldehyde and all paraformaldehyde-based fixations, but only protocol with 4% paraformaldehyde in PHEM buffer allowed accurate imaging of actin filaments, and simultaneously allows monitoring of rickettsiae using actin-based motility during infection inside the host cells.

A case with neurological abnormalities caused by Rickettsia raoultii in northwestern China.

BACKGROUND: The number of new rickettsial species are rapidly increasing, and increasing numbers of Rickettsia raoultii (R. raoultii) infection cases have been detected in humans. However, neurological abnormalities caused by R. raoultii are rarely reported, especially in northwestern China. CASE PRESENTATION: A 36-year-old Kazakh shepherd with an attached tick on part temporalis, presented with right eyelid droop, lethargy, fever, headache, fever (38.0-41.0 °C) and erythematous rash. The examination of cerebrospinal fluid (CSF) showed cerebrospinal pressure of 200 mm H2O, leukocyte count of 300.0 × 106/L, adenosine deaminase of 2.15 U/L, and total protein concentration of 0.93 g/L. The diagnosis of R. raoultii infection was confirmed by six genetic markers, and semi-quantified by enzyme-linked immunosorbent assay for rickettsial antigen. The patient gradually recovered after treatment with doxycycline and ceftriaxone. R. raoultii DNA was found both in a tick detached from this patient and in 0.18% (2/1107) of blood samples collected from local shepherds. CONCLUSIONS: This is the first reported case with neurological abnormalities caused by R. raoultii in northwestern China. It is vital to detect rickettsial agents both in blood and CSF for tick bite patients with neurological abnormalities. Public health workers and physicians should pay attention to neurological abnormalities caused by Rickettsia.

Giant viruses: spore-like missing links between Rickettsia and mitochondria?

Phyloproteomics indicate common viral origin from ancient cells before archaea, bacteria, and eukaryota split and subsequent size and complexity reductions occurred. Further independent evidence for the cellular origin of viruses is reviewed for the virus order Megavirales, focusing on the family Poxviridae. Megavirales comprises giant viruses, double-stranded DNA viruses whose genomes exceed some bacterial ones and large enough to parasitize large-celled protists (amoeba). Giant viruses, virophages, and mitochondria have homologous DNA and RNA polymerases and share RNA splicing punctuation by stem-loop hairpins. Giant virus factories and amoeban mitochondria colocate, with viral proteins homologous to mitochondrial proteins specifically targeting mitochondrial inner membranes. Mitochondria share asexual budding with many bacterial endospores and membrane-enveloped viruses. These megavirus-mitochondrion similarities are not coincidental: systematic alignment analyses have detected candidate megaviral homologs of each amoeban mitogene (including ribosomal RNAs) distributed across megaviral genomes. These candidate megaviral homologs overall follow mitogene order, and megaviral-mitogenome synteny increases with viral genome reduction. This analysis is repeated within Poxviridae, whose organellar-like morphogenesis is reminiscent of mitochondria. More generally, the results confirm the patterns observed in Megavirales: synteny with amoeban mitogenomes increases with genome reduction. Parsimoniously interpreting this suggests Megavirales and mitochondria have a rickettsia-like common ancestor. Megavirales could be the missing links between bacterial-like cells and mitochondria, implying cellular-to-viral-to-subcellular macroevolution.

Wolbachia both aids and hampers the performance of spider mites on different host plants.

In the last few decades, many studies have revealed the potential role of arthropod bacterial endosymbionts in shaping the host range of generalist herbivores and their performance on different host plants, which, in turn, might affect endosymbiont distribution in herbivore populations. We tested this by measuring the prevalence of endosymbionts in natural populations of the generalist spider mite Tetranychus urticae on different host plants. Focusing on Wolbachia, we then analysed how symbionts affected mite life-history traits on the same host plants in the laboratory. Overall, the prevalences of Cardinium and Rickettsia were low, whereas that of Wolbachia was high, with the highest values on bean and eggplant and the lowest on morning glory, tomato and zuchini. Although most mite life-history traits were affected by the plant species only, Wolbachia infection was detrimental for the egg-hatching rate on morning glory and zucchini, and led to a more female-biased sex ratio on morning glory and eggplant. These results suggest that endosymbionts may affect the host range of polyphagous herbivores, both by aiding and hampering their performance, depending on the host plant and on the life-history trait that affects performance the most. Conversely, endosymbiont spread may be facilitated or hindered by the plants on which infected herbivores occur.

A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection.

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

End-in-Sight: Cell Polarization by the Polygamic Organizer PopZ.

Understanding how asymmetries in cellular constituents are achieved and how such positional information directs the construction of structures in a nonrandom fashion is a fundamental problem in cell biology. The recent identification of determinants that self-assemble into macromolecular complexes at the bacterial cell pole provides new insight into the underlying organizational principles in bacterial cells. Specifically, polarity studies in host-associated or free-living α-proteobacteria, a lineage of Gram-negative (diderm) bacteria, reveals that functional and cytological mono- and bipolarity is often conferred by the multivalent polar organizer PopZ, originally identified as a component of a polar chromosome anchor in the cell cycle model system Caulobacter crescentus. PopZ-dependent polarization appears to be widespread and also functional in obligate intracellular pathogens. Here, we discuss how PopZ polarization and the establishment of polar complexes occurs, and we detail the physiological roles of these complexes.

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 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.

Expression of Rickettsia Adr2 protein in E. coli is sufficient to promote resistance to complement-mediated killing, but not adherence to mammalian cells.

Bacteria exposed to host serum are subject to the antibacterial effects to the complement system. However, pathogenic microorganisms have evolved mechanisms of evading this immune attack. We have previously demonstrated that at least two R. conorii antigens, RC1281/Adr1 and OmpB ß-peptide, contribute to the evasion of complement-mediated killing by binding the complement regulatory proteins vitronectin and factor H. RC1282/Adr2, a protein related to Adr1, is predicted to share similar structural features, suggesting that this protein may also contribute to evasion of complement-mediated killing. Interestingly, the R. prowazekii Adr1 and Adr2(RP828) proteins were originally found to interact with host cell surface proteins, suggesting their putative roles as adhesins in this pathogenic rickettsial species. In this study, we expressed both R. conorii and R. prowazekii Adr2 on the surface of a non-adherent, serum-sensitive strain of E. coli to examine the potential role of this protein to mediate evasion of complement-mediated killing and adherence to host cells. We demonstrate that, similar to R. conorii Adr1, R. conorii and R. prowazekii Adr2 are sufficient to mediate serum resistance and to promote interaction with the host complement regulator vitronectin. Furthermore, we demonstrate that expression of Adr2 in a non-adherent strain of E. coli is insufficient to mediate adherence to cultured mammalian endothelial cells. Together, our data demonstrate that the R. conorii and R. prowazekii Adr2 protein does not participate in the interactions with mammalian cells, but rather, participates in the evasion of killing by complement.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria.

Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

Does complement Factor H-Related protein 5 Nephropathy (Troodos Nephropathy) protect from rickettsial infections?

Complement Factor H-Related protein 5 Nephropathy (CFHR5N) is an endemic hereditary renal disease in the island of Cyprus. Although only very recently recognized, it has provided insight into previously unknown genetic aspects of complement-mediated renal diseases and in fact it has contributed to the introduction of the new disease classification, 'C3 Glomerulopathy'. Herein, based on evidence from epidemiological, genetic, clinical and basic research studies, the hypothesis that CFHR5N could be protective from rickettsial infections is proposed. Confirming this hypothesis, could have significant implications for the study of Complement Factor- H Related Proteins (CFHRs) in renal diseases and rickettsial molecular microbiology.

Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.

Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.