Navigating the Med-Peds Maze: Considerations for the Combined Adult and Pediatric Infectious Disease Fellowship Application Process.

Navigating the combined adult and pediatric infectious disease (ID) fellowship application, interview, and matching process requires careful consideration from applicants and programs alike. Currently, it is functional but not streamlined, and as the ID community is facing recruitment and workforce challenges, it is important to be transparent about this process for applicants while emphasizing areas of potential improvement for fellowship programs. As it stands, this process requires foresight from the applicant and coordination between the adult and pediatric fellowship programs. This perspective article provides an anecdote and discusses issues and suggestions for troubleshooting, including, but not limited to: strategic approach to applications, interviews, and ranking, mentorship, geographic preference, and program saturation.

MAIT cell-MR1 reactivity is highly conserved across multiple divergent species.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Potent Immunomodulators Developed from an Unstable Bacterial Metabolite of Vitamin B2 Biosynthesis.

Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17 nM), and their potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.

Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Synthetic 5-amino-6-D-ribitylaminouracil paired with inflammatory stimuli facilitates MAIT cell expansion in vivo.

Introduction: Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which mediate host immunity to microbial infection by recognizing metabolite antigens derived from microbial riboflavin synthesis presented by the MHC-I-related protein 1 (MR1). Namely, the potent MAIT cell antigens, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), form via the condensation of the riboflavin precursor 5-amino-6-D-ribitylaminouracil (5-A-RU) with the reactive carbonyl species (RCS) methylglyoxal (MG) and glyoxal (G), respectively. Although MAIT cells are abundant in humans, they are rare in mice, and increasing their abundance using expansion protocols with antigen and adjuvant has been shown to facilitate their study in mouse models of infection and disease. Methods: Here, we outline three methods to increase the abundance of MAIT cells in C57BL/6 mice using a combination of inflammatory stimuli, 5-A-RU and MG. Results: Our data demonstrate that the administration of synthetic 5-A-RU in combination with one of three different inflammatory stimuli is sufficient to increase the frequency and absolute numbers of MAIT cells in C57BL/6 mice. The resultant boosted MAIT cells are functional and can provide protection against a lethal infection of Legionella longbeachae. Conclusion: These results provide alternative methods for expanding MAIT cells with high doses of commercially available 5-A-RU (± MG) in the presence of various danger signals.

The allopurinol metabolite, oxypurinol, drives oligoclonal expansions of drug-reactive T cells in resolved hypersensitivity cases and drug-naïve healthy donors.

Allopurinol (ALP) is a successful drug used in the treatment of gout. However, this drug has been implicated in hypersensitivity reactions that can cause severe to life-threatening reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Individuals who carry the human leukocyte antigen (HLA)-B*58:01 allotype are at higher risk of experiencing a hypersensitivity reaction (odds ratios ranging from 5.62 to 580.3 for mild to severe reactions, respectively). In addition to the parent drug, the metabolite oxypurinol (OXP) is implicated in triggering T cell-mediated immunopathology via a labile interaction with HLA-B*58:01. To date, there has been limited information regarding the T-cell receptor (TCR) repertoire usage of reactive T cells in patients with ALP-induced SJS or TEN and, in particular, there are no reports examining paired αßTCRs. Here, using in vitro drug-treated PBMCs isolated from both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors, we show that OXP is the driver of CD8+ T cell-mediated responses and that drug-exposed memory T cells can exhibit a proinflammatory immunophenotype similar to T cells described during active disease. Furthermore, this response supported the pharmacological interaction with immune receptors (p-i) concept by showcasing (i) the labile metabolite interaction with peptide/HLA complexes, (ii) immunogenic complex formation at the cell surface, and (iii) lack of requirement for antigen processing to elicit drug-induced T cell responsiveness. Examination of paired OXP-induced αßTCR repertoires highlighted an oligoclonal and private clonotypic profile in both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors.

Molecular insights into metabolite antigen recognition by mucosal-associated invariant T cells.

Metabolite-based T-cell immunity is emerging as a major player in antimicrobial immunity, autoimmunity, and cancer. Here, small-molecule metabolites were identified to be captured and presented by the major histocompatibility complex class-I-related molecule (MR1) to T cells, namely mucosal-associated invariant T cells (MAIT) and diverse MR1-restricted T cells. Both MR1 and MAIT are evolutionarily conserved in many mammals, suggesting important roles in host immunity. Rational chemical modifications of these naturally occurring metabolites, termed altered metabolite ligands (AMLs), have advanced our understanding of the molecular correlates of MAIT T cell receptor (TCR)-MR1 recognition. This review provides a generalized framework for metabolite recognition and modulation of MAIT cells.

Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.

Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.

RIPK3 controls MAIT cell accumulation during development but not during infection.

Cell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.

Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I-related protein 1 MR1.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC50 < 1 µM), moderate (1 µM < IC50 < 100 µM), and weak (IC50 > 100 µM). We demonstrated a clear correlation between ligand-binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed fluorescence polarization-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the A'-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

The establishment of a cytomegalovirus -specific CD8+ T-cell threshold by kinetic modeling for the prediction of post-hemopoietic stem cell transplant reactivation.

The dynamic interaction between the CMV virus and host immune response remains obscure, thus hindering the diagnosis and therapeutic management of patients with HSCT. The current diagnosis of CMV viremia depends on viral load estimation. Medical intervention based on viral load, can be unnecessary or poorly timed for many patients. Here we examined the clinical features and blood samples of patients with HSCT and assessed the CMV reactivation kinetics and corresponding CMV antigen-specific T-cell response in individual patients based on a peptide pool stimulation T-cell assay, which showed that CMV-specific CD8+ T cells were more suitable to be a diagnosis indicator for suppressing CMV reactivation. Using ROC analysis, we defined and verified a CMV-specific CD8+ T-cell counts threshold (925 cells/106 PBMCs) as an indicator of CMV reactivation post-HSCT, and suggested that use of this threshold would provide more accurate guidance for prompt medication and better management of CMV infection post-HSCT.

The role of mucosal-associated invariant T cells in visceral leishmaniasis.

Mucosal-associated invariant T (MAIT) cells are restricted by MR1 and are known to protect against bacterial and viral infections. Our understanding of the role of MAIT cells in parasitic infections, such as visceral leishmaniasis (VL) caused by protozoan parasites of Leishmania donovani, is limited. This study showed that in response to L. infantum, human peripheral blood MAIT cells from children with leishmaniasis produced TNF and IFN-γ in an MR1-dependent manner. The overall frequency of MAIT cells was inversely correlated with alanine aminotransferase levels, a specific marker of liver damage strongly associated with severe hepatic involvement in VL. In addition, there was a positive correlation between total protein levels and the frequency of IL-17A+ CD8+ MAIT cells, whereby reduced total protein levels are a marker of liver and kidney damage. Furthermore, the frequencies of IFN-γ+ and IL-10+ MAIT cells were inversely correlated with hemoglobin levels, a marker of severe anemia. In asymptomatic individuals and VL patients after treatment, MAIT cells also produced IL-17A, a cytokine signature associated with resistance to visceral leishmaniasis, suggesting that MAIT cells play important role in protecting against VL. In summary, these results broaden our understanding of MAIT-cell immunity to include protection against parasitic infections, with implications for MAIT-cell-based therapeutics and vaccines. At last, this study paves the way for the investigation of putative MAIT cell antigens that could exist in the context of Leishmania infection.

CD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells.

Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.

The balance of interleukin-12 and interleukin-23 determines the bias of MAIT1 versus MAIT17 responses during bacterial infection.

Mucosal-associated invariant T (MAIT) cells are a major subset of innate-like T cells mediating protection against bacterial infection through recognition of microbial metabolites derived from riboflavin biosynthesis. Mouse MAIT cells egress from the thymus as two main subpopulations with distinct functions, namely, T-bet-expressing MAIT1 and RORγt-expressing MAIT17 cells. Previously, we reported that inducible T-cell costimulator and interleukin (IL)-23 provide essential signals for optimal MHC-related protein 1 (MR1)-dependent activation and expansion of MAIT17 cells in vivo. Here, in a model of tularemia, in which MAIT1 responses predominate, we demonstrate that IL-12 and IL-23 promote MAIT1 cell expansion during acute infection and that IL-12 is indispensable for MAIT1 phenotype and function. Furthermore, we showed that the bias toward MAIT1 or MAIT17 responses we observed during different bacterial infections was determined and modulated by the balance between IL-12 and IL-23 and that these responses could be recapitulated by cytokine coadministration with antigen. Our results indicate a potential for tailored immunotherapeutic interventions via MAIT cell manipulation.

Dual TCR-α Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRß-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRß-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous ß-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

Differential location of NKT and MAIT cells within lymphoid tissue.

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αß T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

Differential antigenic requirements by diverse MR1-restricted T cells.

MHC-related protein 1 (MR1) presents microbial riboflavin metabolites to mucosal-associated invariant T (MAIT) cells for surveillance of microbial presence. MAIT cells express a semi-invariant T-cell receptor (TCR), which recognizes MR1-antigen complexes in a pattern-recognition-like manner. Recently, diverse populations of MR1-restricted T cells have been described that exhibit broad recognition of tumor cells and appear to recognize MR1 in association with tumor-derived self-antigens, though the identity of these antigens remains unclear. Here, we have used TCR gene transfer and engineered MR1-expressing antigen-presenting cells to probe the MR1 restriction and antigen reactivity of a range of MR1-restricted TCRs, including model tumor-reactive TCRs. We confirm MR1 reactivity by these TCRs, show differential dependence on lysine at position 43 of MR1 (K43) and demonstrate competitive inhibition by the MR1 ligand 6-formylpterin. TCR-expressing reporter lines, however, failed to recapitulate the robust tumor specificity previously reported, suggesting an importance of accessory molecules for MR1-dependent tumor reactivity. Finally, MR1-mutant cell lines showed that distinct residues on the α1/α2 helices were required for TCR binding by different MR1-restricted T cells and suggested central but distinct docking modes by the broad family of MR1-restricted αß TCRs. Collectively, these data are consistent with recognition of distinct antigens by diverse MR1-restricted T cells.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.