Khandra T. Sears

Surface proteome analysis and characterization of surface cell antigen (Sca) or autotransporter family of Rickettsia typhi.

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.

Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells

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.

A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion

ABSTRACT 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.

Functional Characterization of a Phospholipase A2 Homolog from Rickettsia typhi

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.

Reactive oxygen species-dependent destruction of MEK and Akt in Manumycin stimulated death of lymphoid tumor and myeloma cell lines.

Manumycin‐A (Man‐A) is a farnesyltransferase inhibitor (FTI), which was originally identified as an effective tumoricide against several cancers, especially ones harboring constitutively active Ras. However, it is becoming apparent that Man‐A can stimulate tumor death independently of FTases. Antioxidant treatment blocked Man‐A‐stimulated DNA damage and reversed Man‐A‐inhibited tumor growth. However, the precise molecular details of how these reactive oxygen species (ROS) influence cell signaling modules are poorly understood. We examined how ROS may modulate death and survival pathways in a panel of tumor cells. Man‐A treatment resulted in a massive induction of superoxide anion (·O2−) only in Man‐A‐sensitive tumors. Within 1 hr, Man‐A caused the ROS‐dependent activation of caspases 9 and 3. In this time‐frame, the Ras‐Raf target, MEK, and the survival protein Akt were dephosphorylated in ROS‐dependent fashions and then cleaved in ROS and caspase‐dependent manners. Pretreatment with ROS scavengers blocked the adverse effects of Man‐A, including the processing of caspases and the cleavage of MEK and Akt. These events were noted before any losses in Ras activity or changes in its maturation could be detected. Finally, transfection with cDNAs encoding the antioxidant enzymes catalase, superoxide dismutase and thioredoxin reductase inhibited superoxide induction and apoptosis. Together, our data suggest that the elimination of tumors by Man‐A can be independent of the inhibiting of Ras. However, one universal feature observed is the generation of death‐triggering intracellular oxidants that appear to directly participate in the select targeting of growth and survival proteins that then either augment or ensure tumor cell death.

Defending the fort: a role for defensin-2 in limiting Rickettsia montanensis infection of Dermacentor variabilis

The importance of tick defensins is evidenced by their expression in a wide variety of tick tissues and prevalence across many tick genera. To date, the functional and biological significance of defensin‐2 as a rickettsiastatic or rickettsiacidal antimicrobial peptide has not been addressed. In a previous study, defensin‐2 transcription was shown to increase in Dermacentor variabilis ticks challenged with Rickettsia montanensis. In the present study, the hypothesis that defensin‐2 is functional as a rickettsiastatic and/or rickettsiacidal antimicrobial peptide is tested. We show that defensin‐2 plays a role in reducing burden after acquisition of Rickettsia montanensis through capillary feeding. Moreover, defensin‐2 is shown to associate with R. montanensis in vitro and in vivo, causing cytoplasmic leakiness.

Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

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.

Bioactive Immune Components of Anti-Diarrheagenic Enterotoxigenic Escherichia coli Hyperimmune Bovine Colostrum Products

ABSTRACT Diarrhea is a common illness among travelers to resource-limited countries, the most prevalent attributable agent being enterotoxigenic Escherichia coli (ETEC). At this time, there are no vaccines licensed specifically for the prevention of ETEC-induced travelers diarrhea (TD), and this has propelled investigation of alternative preventive methods. Colostrum, the first milk expressed after birthing, is rich in immunoglobulins and innate immune components for protection of newborns against infectious agents. Hyperimmune bovine colostrum (HBC) produced by immunization of cows during gestation (and containing high levels of specific antibodies) is a practical and effective prophylactic tool against gastrointestinal illnesses. A commercial HBC product, Travelan, is available for prevention of ETEC-induced diarrhea. Despite its demonstrated clinical efficacy, the underlying immune components and antimicrobial activity that contribute to protection remain undefined. We investigated innate and adaptive immune components of several commercial HBC products formulated to reduce the risk of ETEC-induced diarrhea, including Travelan and IMM-124E, a newer product that has broader gastrointestinal health benefits. The immune components measured included total and ETEC-specific IgG, total IgA, cytokines, growth factors, and lactoferrin. HBC products contained high levels of IgG specific for multiple ETEC antigens, including O-polysaccharide 78 and colonization factor antigen I (CFA/I) present in the administered vaccines. Antimicrobial activity was measured in vitro using novel functional assays. HBC greatly reduced ETEC motility in soft agar and exhibited bactericidal activity in the presence of complement. We have identified immune components and antimicrobial activity potentially involved in the prevention of ETEC infection by HBC in vivo.

Immunogenicity and protective efficacy against Salmonella C2-C3 infection in mice immunized with a glycoconjugate of S. Newport Core-O polysaccharide linked to the homologous serovar FliC protein

ABSTRACT Nontyphoidal Salmonella (NTS) are important human enteric pathogens globally. Among the different serovars associated with human NTS disease, S. Newport (a serogroup C2-C3 Salmonella) accounts for a measurable proportion of cases. However, to date there are no licensed human NTS vaccines. NTS lipopolysaccharide-associated O polysaccharides are virulence factors and protective antigens in animal models. As isolated molecules, bacterial polysaccharides are generally poorly immunogenic, a limitation overcome by conjugation to a protein carrier. We report herein the development of a candidate serogroup C2-C3 glycoconjugate vaccine based on S. Newport Core-O polysaccharide (COPS) and phase 1 flagellin (FliC). S. Newport COPS and FliC were purified from genetically engineered reagent strains, and conjugated at the polysaccharide reducing end to FliC protein lysines with thioether chemistry. S. Newport COPS:FliC immunization in mice improved anti-polysaccharide immune responses, generated high anti-FliC IgG titers, and mediated robust protection against challenge with both the homologous serovar as well another serogroup C2-C3 serovar (S. Muenchen). Analyses of S. Newport COPS:FliC induced sera found that the anti-COPS IgG antibodies were specific for serogroup C2-C3 lipopolysaccharide, and could promote bactericidal killing by complement and uptake into phagocytes. These preclinical studies establish the protective capacity of serogroup C2-C3 OPS glycoconjugates, and provide a path forward for the development of a multivalent Salmonella vaccine for humans that includes serogroup C2-C3.