Biology subjects

Azad, A. F.

Publications and source records attributed to Azad, A. F..

5 recordsLinked to original sources

bioRxiv2024

Pathogenic rickettsiae utilize the phosphatidylserine binding receptor CD300f on macrophages for host invasion and pathogenesis.

Some arthropod-borne obligate intracellular rickettsiae are among the most virulent human pathogens. Rickettsia species modulate immune (e.g., macrophages; M{Phi}) and non-immune cell (e.g., endothelial cells) responses to create a habitable environment for host colonization. In particular, M{Phi} play a crucial role in either terminating an infection at an early stage or succumbing to bacterial replication and colonization. However, our understanding on how Rickettsia species invade host cells, including M{Phi}, remain poorly defined. In this study, we describe a mechanism of host invasion by Rickettsia species, involving rickettsial phosphatidylserine (PS), as a ligand, and the CD300f receptor on M{Phi}. Using bone marrow-derived macrophages (BMDM{Phi}) from wild-type (WT) and CD300f-/- mice, we demonstrated that engulfment of both pathogenic R. typhi (the etiologic agent of murine typhus) and R. rickettsii (the etiologic agent of Rocky Mountain spotted fever) species as well as the non-pathogenic R. montanensis was significantly reduced in CD300f-/- BMDM{Phi} as compared to that of WT BMDM{Phi}. Furthermore, our mechanistic analysis suggests bacterial PS as the potential source for the CD300f-mediated rickettsiae engulfment by M{Phi}. In vivo infection studies using WT and CD300f-/- C57BL/6J mice showed that CD300f-/- animals were protected against R. typhi-or R. rickettsii-induced fatal rickettsiosis, which correlated with levels of bacterial burden detected in the spleens of mice. Adoptive transfer studies further revealed that CD300f-expressing M{Phi} are important mediators to control rickettsiosis in vivo. Collectively, our findings describe a previously unappreciated role for the efferocytic receptor, CD300f, to facilitate engulfment of rickettsiae within the host.

microbiology↗

bioRxiv2022

Rickettsia typhi peptidoglycan mapping with data-dependent tandem mass spectrometry

Rickettsia species are diverse Gram-negative obligate intracellular bacteria often pervasive in numerous invertebrates, as well as fungal, nematode and microeukaryotic hosts. Certain species are etiological agents for well-known arthropod-borne illnesses; e.g., R. rickettsii (Rocky Mountain Spotted Fever), R. prowazekii (Epidemic Typhus), and R. typhi, (Endemic Typhus). Living freely in eukaryotic cytosol presumably exposes rickettsiae to host cell immune receptors, particularly those recognizing bacterial cell envelope glycoconjugates. However, the mechanics of host recognition of rickettsiae remain poorly defined. As rickettsiae synthesize a canonical Gram-negative cell envelope that includes peptidoglycan (PGN) and lipopolysaccharide (LPS), structural insight on these macromolecules is important for deciphering host responses to these pathogens. In this work, PGN from R. typhi was digested and the resultant subunits were analyzed by two different, albeit complementary, sample preparation methods. Both approaches were subsequently subjected to liquid chromatography/mass spectrometry analysis to infer PGN structure. R. typhi PGN was determined to be similar to most other Gram-negative bacteria, with mDAP-type muropeptide subunits. However, additional alanine residues were observed elongating the muropeptide stems, rather than the glycine residues usually observed in Gram-negative bacterial PGN. Despite this deviation, R. typhi contains a murein layer that is predicted to agonize host cellular PGN receptors and be susceptible to PGN-targeting antimicrobials. This same structure is likely synthesized by all Rickettsia species, as bioinformatics and comparative genomics analyses indicate the biosynthesis of PGN is highly conserved. Determining how host cells process this canonical glycoconjugate during infection is crucial for identifying factors behind rickettsial pathogenesis, including immunoavoidance or proinflammatory mechanisms possibly employed by rickettsiae with varying pathogenic potential.

microbiology↗

bioRxiv2021

Pathogenic, but not non-pathogenic, Rickettsia manipulate inflammasome-dependent IL-1 responses to facilitate their replication and host dissemination.

Rickettsia species (spp.) are strict obligate intracellular bacteria, with some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis are evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remains elusive. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected with R. typhi (etiologic agent of murine typhus) and R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenic R. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1 and to a lesser extent IL- 1{beta}. Antibody-mediated neutralization of IL-1 confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infected WT mice. As macrophages are a primary source of both IL-1 and IL-1{beta} cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis, eluded pro- IL-1 induction and benefited pre-dominantly from the reduced IL-1 secretion, via a Caspase-11-Gsdmd-dependent pathway, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1 secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1 signaling axis in macrophages thus supporting their replication within the host. IMPORTANCECurrently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1 signaling axis, to limiting the replication of pathogenic R. rickettsia and R. typhi species in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptative transfer studies further identified IL-1-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenic Rickettsia spp., benefit from a reduction in the Caspase-11-Gsdmd-mediated release of IL-1 to support host colonization.

microbiology↗

bioRxiv2020

Evolution of Wolbachia Mutualism and Reproductive Parasitism: Insight from Two Novel Strains that Co-infect Cat Fleas

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA). wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the Wolbachia tree, indicating parallel evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon analogous to the wPip cinAB operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and immediate-5 end genes are syntenic to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of larger toxins. Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though occasionally wCfeJ singly infects fleas in wild populations. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism. ImportanceMany arthropod and certain nematode species are infected with wolbachiae which are intracellular bacteria well known for reproductive parasitism (RP). Like other RP strategies, Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and frequency in host populations. Mutualism is another strategy employed by wolbachiae to maintain host infection, with some strains synthesizing and supplementing certain B vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novel Wolbachia strains that co-infect cat fleas (Ctenocephalides felis): wCfeT carries biotin synthesis genes, while wCfeJ carries a CI-inducing toxin-antidote operon. Our analyses of these genes highlight their mobility across the Wolbachia phylogeny and source to other intracellular bacteria. Remarkably, the C. felis genome also carries two CI-like antidote genes divergent from the wCfeJ antidote gene, indicating episodic RP in cat fleas. Collectively, wCfeT and wCfeJ inform on the rampant dissemination of diverse factors that mediate Wolbachia strategies for persisting in invertebrate host populations.

genomics↗

bioRxiv2020

Cat fleas in flux: Rampant gene duplication, genome size plasticity, and paradoxical Wolbachia infection

BackgroundFleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e. the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. ResultsBy combining Illumina and PacBio sequencing with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by [~]38% of protein-coding genes with two or more copies and over 4,000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable "genome-in-flux" phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. ConclusionRampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts - potential tools for blocking pathogen transmission - these oddities highlight a unique and underappreciated disease vector.

genomics↗