The recently described rickettsial protein RoaM (regulator of actin-based motility) negatively regulates the production of actin tails, and its abrogation induces hyper-spreading behavior in many laboratory-adapted strains of Rickettsia rickettsii. RoaM is not surface exposed; thus, its mechanism of regulating actin-based motility is unclear. Using R. rickettsii strains derived from the virulent Sheila Smith strain that express varying levels of roaM, an RNA-seq experiment was performed. We found that roaM-overexpressing strains downregulate expression of at least six genes which may link the regulatory effects of RoaM to the phenotypic effect on motility. Genes regulated by RoaM were confirmed by RT-qPCR. Among the genes regulated is the secreted effector RarP2, which disrupts the trans-Golgi network. Two of the hypothetical proteins were shown to be secreted via fusion to a glycogen synthase kinase tag, which when phosphorylated reveals exposure to the host-cell cytosol. Taken together, these data support the hypothesis that RoaM affects transcription, downregulating rickettsial genes important for pathogenicity in the mammalian host but which are perhaps otherwise detrimental within the tick vector. To determine how RoaM activity may itself be regulated, we investigated a role of temperature in roaM transcription. RoaM expression itself is not temperature dependent, but many other rickettsial genes are, including some also regulated by RoaM. This suggests that rickettsiae utilize multiple mechanisms to control gene expression in response to environmental signals. IMPORTANCE:RoaM was previously shown to repress the production of actin tails by unknown mechanisms. The roaM gene is negatively selected for in cell culture resulting in hyper-spreading mutants. This work reveals that rather than specifically regulating motility in Rickettsia rickettsii, a set of rickettsial genes is downregulated that includes the type IV secreted effector, rarP2, as well as two other secreted, putative effectors. Relatively few secreted effectors have been identified in Rickettsia. RoaM appears to be part of a larger biological program encompassing active spreading in mammalian cells and may be a critical component for R. rickettsii to transition from arthropod to mammalian host.

Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.

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

Members of the spotted fever group rickettsia express four large, surface-exposed autotransporters, at least one of which is a known virulence determinant. Autotransporter translocation to the bacterial outer surface, also known as type V secretion, involves formation of a β-barrel autotransporter domain in the periplasm that inserts into the outer membrane to form a pore through which the N-terminal passenger domain is passed and exposed on the outer surface. Two major surface antigens of Rickettsia rickettsii, are known to be surface exposed and the passenger domain cleaved from the autotransporter domain. A highly passaged strain of R. rickettsii, Iowa, fails to cleave these autotransporters and is avirulent. We have identified a putative peptidase, truncated in the Iowa strain, that when reconstituted into Iowa restores appropriate processing of the autotransporters as well as restoring a modest degree of virulence.

Complete genomes of Rickettsia rickettsii were sequenced with Illumina and PacBio technologies from low-passage isolates from ticks. These isolates were quality controlled for intact roaM, a regulator of actin-based motility that is negatively selected for in culture. The Sheila Smith strain was re-sequenced using the same methodology.

Mitochondria are critical cellular organelles that perform a wide variety of functions including energy production and immune regulation. To perform these functions, mitochondria contain approximately 1,500 proteins, the majority of which are encoded in the nuclear genome, translated in the cytoplasm, and translocated to the mitochondria using distinct mitochondria targeting sequences (MTS). Bacterial proteins can also contain MTS and localize to the mitochondria. For the obligate intracellular human pathogen, Chlamydia trachomatis , interaction with various host cell organelles promotes intracellular replication. However, the extent and mechanisms through which Chlamydia interact directly with mitochondria remain unclear. We investigated the presence of MTS in the C. trachomatis genome and discovered 30 genes with around 70% or greater probability of mitochondrial localization. Five are translocated to the mitochondria upon ectopic expression in HeLa cells. Mass spectrometry of isolated mitochondria from infected cells revealed that two of these proteins localize to the mitochondria during infection. Comparison of mitochondria from infected and uninfected cells suggests that chlamydial infection affects mitochondrial protein composition. Around 125 host proteins were significantly decreased or absent in mitochondria from infected cells. Among these are pro-apoptotic factors and those related to mitochondrial fission/fusion dynamics. Conversely, 82 host proteins were increased in or specific to mitochondria of infected cells, many of which act as anti-apoptotic factors and upregulators of cellular metabolism. These data support the notion that C. trachomatis specifically targets host mitochondria to manipulate cell fate decisions and metabolic function to support pathogen survival and replication. Importance Obligate intracellular bacteria have evolved multiple means to promote their intracellular survival and replication within the otherwise harsh environment of the eukaryotic cell. Nutrient acquisition and avoidance of cellular defense mechanisms are critical to an intracellular lifestyle. Mitochondria are critical organelles that produce energy in the form of ATP and regulate programmed cell death responses to invasive pathogenic microbes. Cell death prior to completion of replication would be detrimental to the pathogen. C. trachomatis produces at least two and possibly more proteins that target the mitochondria. Collectively, C. trachomatis infection modulates mitochondrial protein composition favoring a profile suggestive of down-regulation of apoptosis.

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.

Obligate intracellular bacteria have evolved multiple means to promote their intracellular survival and replication within the otherwise harsh environment of the eukaryotic cell. Nutrient acquisition and avoidance of cellular defense mechanisms are critical to an intracellular lifestyle.

The obligate intracellular bacterium, Chlamydia trachomatis, replicates within a parasitophorous vacuole termed an inclusion. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate, intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible NO synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii. Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii cells were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent antirickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Fragmentation of the Golgi apparatus is observed during a number of physiological processes including mitosis and apoptosis, but also occurs in pathological states such as neurodegenerative diseases and some infectious diseases. Here we show that highly virulent strains of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, induce selective fragmentation of the trans-Golgi network (TGN) soon after infection of host cells by secretion of the effector protein Rickettsial Ankyrin Repeat Protein 2 (RARP2). Remarkably, this fragmentation is pronounced for the trans-Golgi network but the cis-Golgi remains largely intact and appropriately localized. Thus R. rickettsii targets specifically the TGN and not the entire Golgi apparatus. Dispersal of the TGN is mediated by the secreted effector protein RARP2, a recently identified type IV secreted effector that is a member of the clan CD cysteine proteases. Site-directed mutagenesis of a predicted cysteine protease active site in RARP2 prevents TGN disruption. General protein transport to the cell surface is severely impacted in cells infected with virulent strains of R. rickettsii. These findings suggest a novel manipulation of cellular organization by an obligate intracellular bacterium to determine interactions with the host cell.

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of human infections. Chlamydiae have a biphasic developmental cycle that takes place within a membrane bound compartment termed an inclusion. After replication in the host cell, the chlamydiae are released by lysis of the host cell or through a process by which the inclusion is extruded [1]. The extrusion process is believed to be controlled by small protein microdomains embedded in the inclusion membrane. For that reason, we sought to image these microdomains of inclusion membrane proteins (Incs) to elucidate the mechanism of chlamydial release [2,3].

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection.

ABSTRACT 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.

Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726–825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α-helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726–825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α-helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins.

Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a vacuole termed an inclusion. At the end of their intracellular developmental cycle, chlamydiae are released either by lysis of the host cell or extrusion of the intact inclusion. The inclusion membrane is extensively modified by the insertion of type III secreted inclusion membrane proteins, Incs, which contribute to inclusion membrane structure and facilitate host-pathogen interactions. An interaction was identified between the inclusion membrane protein, MrcA, and the Ca2+ channel inositol-1,4,5-trisphosphate receptor, type 3 (ITPR3). ITPR3 was recruited and localized to active Src-family-kinase rich microdomains on the inclusion membrane as was the Ca2+ sensor, STIM1. Disruption of MrcA by directed mutagenesis resulted in loss of ITPR3 recruitment and simultaneous reduction of chlamydial release by extrusion. Complementation of MrcA restored ITPR3 recruitment and extrusion. Inhibition of extrusion was also observed following siRNA depletion of host ITPR3 or STIM1. Chlamydial extrusion was also inhibited by the calcium chelator BAPTA-AM. Each of these treatments resulted in a concomitant reduction in phosphorylation of the myosin regulatory light chain (MLC2) and a loss of myosin motor activity at the end of the developmental cycle which is consistent with the reduced extrusion formation. These studies suggest that Ca2+ signaling pathways play an important role in regulation of release mechanisms by C. trachomatis.

ABSTRACT Bacteria of the genus Chlamydia include the significant human pathogens Chlamydia trachomatis and C. pneumoniae. All chlamydiae are obligate intracellular parasites that depend on infection of a host cell and transition through a biphasic developmental cycle. Following host cell invasion by the infectious elementary body (EB), the pathogen transitions to the replicative but noninfectious reticulate body (RB). Differentiation of the RB back to the EB is essential to generate infectious progeny. While the EB form has historically been regarded as metabolically inert, maintenance of infectivity during incubation with specific nutrients has revealed active maintenance of the infectious phenotype. Using transcriptome sequencing, we show that the transcriptome of extracellular EBs incubated under metabolically stimulating conditions does not cluster with germinating EBs but rather with the transcriptome of EBs isolated directly from infected cells. In addition, the transcriptional profile of the extracellular metabolizing EBs more closely resembled that of EB production than germination. Maintenance of infectivity of extracellular EBs was achieved by metabolizing chemically diverse compounds, including glucose 6-phosphate, ATP, and amino acids, all of which can be found in extracellular environments, including mucosal secretions. We further show that the EB cell type actively maintains infectivity in the inclusion after terminal differentiation. Overall, these findings contribute to the emerging understanding that the EB cell form is actively maintained through metabolic processes after terminal differentiation to facilitate prolonged infectivity within the inclusion and under host cell free conditions, for example, following deposition at mucosal surfaces. IMPORTANCE Chlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a wide range of diseases in both animal and human hosts. According to the Centers for Disease Control and Prevention, C. trachomatis is the most frequently reported sexually transmitted infection in the United States, costing the American health care system nearly $2.4 billion annually. Every year, there are over 4 million new cases of Chlamydia infections in the United States and an estimated 100 million cases worldwide. To cause disease, Chlamydia must successfully complete its complex biphasic developmental cycle, alternating between an infectious cell form (EB) specialized for initiating entry into target cells and a replicative form (RB) specialized for creating and maintaining the intracellular replication niche. The EB cell form has historically been considered metabolically quiescent, a passive entity simply waiting for contact with a host cell to initiate the next round of infection. Recent studies and data presented here demonstrate that the EB maintains its infectious phenotype by actively metabolizing a variety of nutrients. Therefore, the EB appears to have an active role in chlamydial biology, possibly within multiple environments, such as mucosal surfaces, fomites, and inside the host cell after formation.