S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.     

Characterization and growth of polymorphic Rickettsia felis in a tick cell line

Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.     

The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.     

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.     

Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.     

Sec24 interaction is essential for localization and virulence-associated function of the bacterial effector protein NleA

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease.     

A new twist on an old pathway--accessory Sec [corrected] systems

The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria, there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria - the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-β, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-β, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-β activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-β-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

On the nature of obligate intracellular symbiosis of rickettsiae — Rickettsia prowazekii cells import mitochondrial porin

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.     

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.     

Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae

Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.