Early career Latinas in STEM: Challenges and solutions.

Mexican, Puerto Rican, and Central American Ancestry (MPRCA) individuals represent 82% of US Latinos. An intergenerational group of MPRCA women and allies met to discuss persistent underrepresentation of MPRCA women in STEM, identifying multi-level challenges and solutions. Implementation of these solutions is important and will benefit MPRCA women and the entire academic community.

Trichomonas vaginalis extracellular vesicles up-regulate and directly transfer adherence factors promoting host cell colonization.

Trichomonas vaginalis, a common sexually transmitted parasite that colonizes the human urogenital tract, secretes extracellular vesicles (TvEVs) that are taken up by human cells and are speculated to be taken up by parasites as well. While the crosstalk between TvEVs and human cells has led to insight into host:parasite interactions, roles for TvEVs in infection have largely been one-sided, with little known about the effect of TvEV uptake by T. vaginalis. Approximately 11% of infections are found to be coinfections of multiple T. vaginalis strains. Clinical isolates often differ in their adherence to and cytolysis of host cells, underscoring the importance of understanding the effects of TvEV uptake within the parasite population. To address this question, our lab tested the ability of a less adherent strain of T. vaginalis, G3, to take up fluorescently labeled TvEVs derived from both itself (G3-EVs) and TvEVs from a more adherent strain of the parasite (B7RC2-EVs). Here, we showed that TvEVs generated from the more adherent strain are internalized more efficiently compared to the less adherent strain. Additionally, preincubation of G3 parasites with B7RC2-EVs increases parasite aggregation and adherence to host cells. Transcriptomics revealed that TvEVs up-regulate expression of predicted parasite membrane proteins and identified an adherence factor, heteropolysaccharide binding protein (HPB2). Finally, using comparative proteomics and superresolution microscopy, we demonstrated direct transfer of an adherence factor, cadherin-like protein, from TvEVs to the recipient parasite's surface. This work identifies TvEVs as a mediator of parasite:parasite communication that may impact pathogenesis during mixed infections.

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.

The murine vaginal microbiota and its perturbation by the human pathogen group B Streptococcus.

BACKGROUND: Composition of the vaginal microbiota has significant influence on female urogenital health and control of infectious disease. Murine models are widely utilized to characterize host-pathogen interactions within the vaginal tract, however, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. Here, we employ 16S rRNA amplicon sequencing to establish the native microbial composition of the vaginal tract in adult C57Bl/6 J mice. We further interrogate the impact of estrous cycle and introduction of the human vaginal pathobiont, group B Streptococcus (GBS) on community state type and stability, and conversely, the impact of the vaginal microbiota on GBS persistence. RESULTS: Sequencing analysis revealed five distinctive community states of the vaginal microbiota dominated largely by Staphylococcus and/or Enterococcus, Lactobacillus, or a mixed population. Stage of estrus did not impact microbial composition. Introduction of GBS decreased community stability at early timepoints; and in some mice, GBS became the dominant bacterium by day 21. Endogenous Staphylococcus abundance correlated with GBS ascension into the uterus, and increased community stability in GBS-challenged mice. CONCLUSIONS: The murine vaginal flora is diverse and fluctuates independently of the estrous cycle. Endogenous flora may impact pathogen colonization and dissemination and should be considered in urogenital infection models.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells.

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.

Insect stage-specific receptor adenylate cyclases are localized to distinct subdomains of the Trypanosoma brucei Flagellar membrane.

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.

Reversible association of tetraspanin with Trichomonas vaginalis flagella upon adherence to host cells.

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analyses of T. vaginalis tetraspanin 6 (TvTSP6). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We observed that TvTSP6 expression is upregulated upon contact with vaginal ectocervical cells (VECs) and that parasite strains that are highly adherent to VECs express higher levels of TvTSP6 mRNA relative to poorly adherent strains. TvTSP6 is localized predominantly on the flagella of parasites cultured in the absence of host cells; however, adherence of the parasite to VECs initially results in a redistribution of the protein to intracellular vesicles and the plasma membrane of the main body of the cell. We found that a 16-amino-acid C-terminal intracellular tail of TvTSP6 is necessary and sufficient for flagellar localization and protein redistribution when the parasite is in contact with VECs. Additionally, deletion of the C-terminal tail reduced parasite migration through Matrigel, a mimic of the extracellular matrix. Together, our data support roles for TvTSP6 in parasite migration in the host and sensory reception during infection.

Unrecognized Potent Activities of Colistin Against Clinically Important mcr+ Enterobacteriaceae Revealed in Synergy with Host Immunity.

Colistin (COL) is a cationic cyclic peptide that disrupts negatively-charged bacterial cell membranes and frequently serves as an antibiotic of last resort to combat multidrug-resistant Gram-negative bacterial infections. Emergence of the horizontally transferable plasmid-borne mobilized colistin resistance (mcr) determinant and its spread to Gram-negative strains harboring extended-spectrum ß-lactamase and carbapenemase resistance genes threatens futility of our chemotherapeutic arsenal. COL is widely regarded to have zero activity against mcr+ patients based on standard antimicrobial susceptibility testing (AST) performed in enriched bacteriological growth media; consequently, the drug is withheld from patients with mcr+ infections. However, these standard testing media poorly mimic in vivo physiology and omit host immune factors. Here we report previously unrecognized bactericidal activities of COL against mcr-1+ isolates of Escherichia coli (EC), Klebsiella pneumoniae (KP), and Salmonella enterica (SE) in standard tissue culture media containing the physiological buffer bicarbonate. Moreover, COL promoted serum complement deposition on the mcr-1+ Gram-negative bacterial surface and synergized potently with active human serum in pathogen killing. At COL concentrations readily achievable with standard dosing, the peptide antibiotic killed mcr-1+ EC, KP, and SE in freshly isolated human blood proved effective as monotherapy in a murine model of mcr-1+ EC bacteremia. Our results suggest that COL, currently ignored as a treatment option based on traditional AST, may in fact benefit patients with mcr-1+ Gram negative infections based on evaluations performed in a more physiologic context. These concepts warrant careful consideration in the clinical microbiology laboratory and for future clinical investigation of their merits in high risk patients with limited therapeutic options.

Mutually exclusive genotypes for pyrazinamide and 5-chloropyrazinamide resistance reveal a potential resistance-proofing strategy.

The pyrazinamide (PZA) analog 5-chloropyrazinamide (5-Cl PZA) is active against mycobacterial species, including PZA-resistant strains of Mycobacterium tuberculosis. In M. smegmatis, overexpression of the type 1 fatty acid synthase (FAS I) confers resistance to 5-Cl PZA, a potent FAS I inhibitor. Since M. tuberculosis and M. bovis cannot tolerate FAS I overexpression, 5-Cl PZA resistance mutations have yet to be described for tubercle bacilli. In an attempt to identify other factors that govern the activity of 5-Cl PZA, we selected for 5-Cl PZA-resistant isolates from a library of transposon-mutagenized M. smegmatis isolates. Here, we report that increased expression of the M. smegmatis pyrazinamidase PzaA confers resistance to 5-Cl PZA and susceptibility to PZA in M. smegmatis, M. tuberculosis, and M. bovis. In contrast, while ectopic overexpression of the M. tuberculosis pyrazinamidase PncA increases PZA susceptibility, this amidase does not mediate resistance to 5-Cl PZA. We conclude that PncA-independent turnover of 5-Cl PZA represents a potential mechanism of resistance to this compound for M. tuberculosis, which will likely translate into enhanced PZA susceptibility. Thus, countersusceptibility can be manipulated as a resistance-proofing strategy for PZA-based compounds when these agents are used simultaneously.

Dynamic Interactions of Group A Streptococcus with Host Macrophages.

Macrophages play a critical role in Group A Streptococcus (GAS) recognition and the consequent activation of innate immunity and inflammatory responses against the pathogen. In parallel, GAS deploys several strategies for escaping detection and elimination by these efficient phagocytic cells. The events that take place in this GAS-macrophage battleground, the cellular consequences for the pathogen and for the immune cell, and the balance between the magnitude of infection and the efficiency of the host immune response can be investigated with a variety of assays presented in this chapter.

Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1ß (IL-1ß). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1ß signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1ß release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1ß release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1ß secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1ß production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1ß expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

Targeting the Achievement Gap: Strategies Toward Removing Inequities in Undergraduate Immunology Education.

A diverse student body enriches the classroom with lived experiences, varied skillsets, community and cultural knowledge, resiliency, and altruistic interests, all critical attributes that benefit both the classroom and the STEM field at large. However, a persistent disparity in academic and educational attainment exists between under-represented minority (URM) and non-URM students in STEM fields. This achievement gap discourages talented URM students from entering STEM professions, threatening the potential, expertise, and perspective of these professions. Here we describe the factors that contribute to the achievement gap and present strategies, utilized in our Immunology classrooms, for combating each factor. We discuss project-based learning pedagogy to give students increased agency and feelings of empowerment. We also highlight concrete practices to foster students' science identities and sense of community, factors that highly promote STEM retention. The dynamic subject of Immunology provides myriad opportunities to implement a curriculum committed to equity, as we outline below.

A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis.

Trichomonas vaginalis, a prevalent sexually transmitted parasite, adheres to and induces cytolysis of human mucosal epithelial cells. We have characterized a hypothetical protein, TVAG_393390, with predicted tertiary structure similar to that of mammalian cadherin proteins involved in cell-cell adherence. TVAG_393390, renamed cadherin-like protein (CLP), contains a calcium-binding site at a position conserved in cadherins. CLP is surface localized, and its mRNA and protein levels are significantly upregulated upon parasite adherence to host cells. To test the roles of CLP and its calcium-binding dependency during host cell adherence, we first demonstrated that wild-type CLP (CLP) binds calcium with a high affinity, whereas the calcium-binding site mutant protein (CLP-mut) does not. CLP and CLP-mut constructs were then used to overexpress these proteins in T. vaginalis Parasites overexpressing CLP have ∼3.5-fold greater adherence to host cells than wild-type parasites, and this increased adherence is ablated by mutating the calcium-binding site. Additionally, competition with recombinant CLP decreased parasite binding to host cells. We also found that overexpression of CLP induced parasite aggregation which was further enhanced in the presence of calcium, whereas CLP-mut overexpression did not affect aggregation. Lastly, parasites overexpressing wild-type CLP induced killing of host cells ∼2.35-fold, whereas parasites overexpressing CLP-mut did not have this effect. These analyses describe the first parasitic CLP and demonstrate a role for this protein in mediating parasite-parasite and host-parasite interactions. T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis.IMPORTANCE The adherence of pathogens to host cells is critical for colonization of the host and establishing infection. Here we identify a protein with no known function that is more abundant on the surface of parasites that are better at binding host cells. To interrogate a predicted function of this protein, we utilized bioinformatic protein prediction programs which allowed us to uncover the first cadherin-like protein (CLP) found in a parasite. Cadherin proteins are conserved metazoan proteins with central roles in cell-cell adhesion, development, and tissue structure maintenance. Functional characterization of this CLP from the unicellular parasite Trichomonas vaginalis demonstrated that the protein mediates both parasite-parasite and parasite-host adherence, which leads to an enhanced killing of host cells by T. vaginalis Our findings demonstrate the presence of CLPs in unicellular pathogens and identify a new host cell binding protein family in a human-infective parasite.

The Fungal Pathogen Candida albicans Promotes Bladder Colonization of Group B Streptococcus.

Group B Streptococcus (GBS) is a common cause of bacterial urinary tract infections (UTI) in susceptible populations, including pregnant women and the elderly. However, the factors that govern GBS persistence and disease severity in this niche are not fully understood. Here, we report that the presence of the fungus Candida albicans, a common urogenital colonizer, can promote GBS UTI. Co-inoculation of GBS with C. albicans increased bacterial adherence to bladder epithelium and promoted GBS colonization in vivo in a C. albicans adhesin-dependent manner. This study demonstrates that fungal colonization of the urogenital tract may be an important determinant of bacterial pathogenesis during UTI.

Trichomonas vaginalis Induces NLRP3 Inflammasome Activation and Pyroptotic Cell Death in Human Macrophages.

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1ß to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1ß production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1ß cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

Group A streptococcal M protein activates the NLRP3 inflammasome.

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1ß (IL-1ß) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1ß activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1ß release could aggravate tissue injury.