Biology subjects

Salazar, J. C.

Publications and source records attributed to Salazar, J. C..

5 recordsLinked to original sources

bioRxiv2025

Sequence variability of BamA and FadL candidate vaccinogens suggests divergent evolutionary paths of Treponema pallidum outer membrane proteins

Knowledge of Treponema pallidum subspecies pallidum (TPA) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols-, three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Lastly, we noted that BamA and FadL candidate ECL vaccinogens previously shown to elicit antibodies that kill treponemes during in vitro cultivation are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing selection of vaccine targets. IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

microbiology↗

bioRxiv2022

High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny

Sequencing of most Treponema pallidum (T. pallidum) genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modelling of TP0470 suggested that the eight residue repeats form an extended -helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged {beta}- sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

microbiology↗

bioRxiv2019

Immunization of mice with chimeric antigens displaying selected epitopes confers protection against intestinal colonization and renal damage caused by Shiga toxin-producing Escherichia coli (STEC).

Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7 and the intranasal conferred protection against renal damage caused by STEC O91:H21. This pre-clinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections.

microbiology↗

bioRxiv2019

Macrophage mediated recognition and clearance of Borrelia burgdorferi elicits MyD88-dependent and - independent phagosomal signals that contribute to phagocytosis and inflammation

Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi (Bb). It is believed that the robust inflammatory response induced by the hosts innate immune system is responsible for the clinical manifestations associated with Bb infection. The macrophage plays a central role in the immune response to many bacterial infections and is thought to play a central role in activation of the innate immune response to Bb. Previous studies have shown that following phagocytosis of spirochetes by macrophages, phagosome maturation results in degradation of Bb and liberation of bacterial lipoproteins and nucleic acids, which are recognized by TLR2 and TLR8, respectively, and elicit MyD88-mediated phagosome signaling cascades. Bone marrow-derived macrophages (BMDMs) from MyD88-/- mice show significantly reduced spirochete uptake and inflammatory cytokine production when incubated with Bb ex vivo. Paradoxically, additional studies revealed that Bb-infected MyD88-/- mice exhibit inflammation in joint and heart tissues. To determine the contribution of MyD88 to macrophage-mediated spirochete clearance, we compared wildtype (WT) and MyD88-/- mice using a murine model of Lyme disease. MyD88-/- mice showed increased Bb burdens in hearts 28 days post infection, while H&E staining and immunohistochemistry showed significantly increased inflammation and greater macrophage infiltrate in the hearts of MyD88-/- mice. This suggests that Bb triggers MyD88-independent inflammatory pathways in macrophages to facilitate cell recruitment to tissues. Upon stimulation with Bb ex vivo, WT and MyD88-/- BMDMs exhibit significant differences in bacteria uptake, suggesting that MyD88 signaling mediates cytoskeleton remodeling and the formation of membrane protrusions to enhance bacteria phagocytosis. A comprehensive transcriptome comparison in Bb-infected WT and MyD88-/- BMDMs identified a large cohort of MyD88-dependent genes that are differentially expressed in response to Bb, including genes involved in actin and cytoskeleton organization (Daam1, Fmnl1). We also identified a cohort of differentially-expressed MyD88-independent chemokines (Cxcl2, Ccl9) known to recruit macrophages. We identified master regulators and generated networks which model potential signaling pathways that mediate both phagocytosis and the inflammatory response. These data provide strong evidence that MyD88-dependent and -independent phagosomal signaling cascades in macrophages play significant roles in the ability of these cells to phagocytose Bb and mediate infection.\n\nAUTHOR SUMMARYMacrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied Bb-infected wildtype (WT) and MyD88-/- mice and Bb-stimulated bone marrow-derived macrophages (BMDMs). Bb-infected MyD88-/- mice show increased bacterial burdens, macrophage infiltration and altered gene expression in inflamed heart tissue. MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes involved in re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-independent master regulators (Cxcl2 and Vcam1) and MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. These results provide mechanistic insights into MyD88-mediated phagosomal signaling enhancing Bb uptake and clearance.

immunology↗

bioRxiv2018

Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of Treponema pallidum subsp. pallidum, the syphilis spirochete.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-exposed {beta}-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of {beta}-barrel-encoding regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) {beta}-barrels in clinical strains could be grouped according to allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored {beta}-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane {beta}-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive TPA OMP diversity and that genetic exchange contributes to the evolutionary biology of TPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.\n\nIMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum (TPA), little is known about how their surface-exposed, {beta}-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the {beta}-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in TPA from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within {beta}-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations drive TPA OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of TPA and frame strategies for vaccine development based upon conserved OMP extracellular loops.

microbiology↗