Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion.

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

TolC-dependent secretion of an ankyrin repeat-containing protein of Rickettsia typhi.

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

Rapid selection of single-stranded DNA aptamers binding Staphylococcus epidermidis in platelet concentrates.

Staphylococcus epidermidis is the most common transfusion-associated pathogen contaminating platelet concentrates. Methods to reduce or eliminate contaminating bacteria from platelet units are critical for improving the safety of blood transfusions. We used rapid isolation of DNA aptamers (RIDA) to identify single-stranded (ss)DNA aptamers as ligands that specifically bind to S. epidermidis. Five target-specific ssDNA aptamers (76 mer) were obtained under stringent selection conditions. Aptamer SE43 demonstrated higher binding affinity compared with scrambled control. Furthermore, when binding assays were conducted in platelet concentrate, there was a twofold increase in binding affinity compared with the SE43 binding in buffer alone. Our data identified an aptamer that may be useful as a ligand to capture, detect or remove S. epidermidis contaminant from platelet concentrates.

Mutant SOD1 mediated pathogenesis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neural disorder that causes death of the motor neurons in the brain and spinal cord; this affects the voluntary muscles and gradually leads to paralysis of the whole body. Most ALS cases are sporadic, though about 5-10% are familial. ALS is caused by multiple factors including mutation in any one of a number of specific genes, one of the most frequently affected is superoxide dismutase (SOD) 1. Alterations in SOD 1 have been linked with several variants of familial ALS. SOD 1 is a powerful antioxidant enzyme that protects cells from the damaging effects of superoxide radicals. The enzyme binds both copper and zinc ions that are directly involved in the deactivation of toxic superoxide radicals. Mutated SOD1 gene can acquire both gain and loss of function mutations. The most commonly identified mutations in SOD1 that affect protein activity are D90A, A4V and G93A. Deleterious mutations have been shown to modify SOD1 activity, which leads to the accumulation of highly toxic hydroxyl radicals. Accumulation of these free radicals causes degradation of both nuclear and mitochondrial DNA and protein misfolding, features which can be used as pathological indicators associated with ALS. Numerous clinical trials have been carried out over last few years with limited success. In some patients advanced techniques like gene and stem cell therapy have been trialed. However no definitive treatment option can provide a cure and currently ALS is managed by drugs and other supportive therapies. Consequently there is a need to identify new approaches for treatment of this ultimately fatal disease.

Secretome of obligate intracellular Rickettsia.

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

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.