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Abstract Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.

ABSTRACT Diffuse panbronchiolitis (DPB) is a rare, idiopathic inflammatory disease primarily diagnosed in East Asian populations. DPB is characterized by diffuse pulmonary lesions, inflammation of the respiratory bronchioles, and bacterial infections of the airway. Historically, sputum cultures reveal Pseudomonas aeruginosa in 22% of DPB patients, increasing to 60% after 4 years from disease onset. Although DPB patients have a known susceptibility to respiratory P. aeruginosa infections, as is observed in other chronic lung diseases such as cystic fibrosis (CF), the characterization of DPB P. aeruginosa strains is limited. In this study, we characterized 24 strains obtained from a cohort of DPB patients for traits previously associated with virulence, including growth, motility, antibiotic susceptibility, lipopolysaccharide structure, and genomic diversity. Our cohort of DPB P. aeruginosa strains exhibits considerable genomic variability when compared with isolates from people with cystic fibrosis chronically colonized with P. aeruginosa and acute P. aeruginosa infection isolates. Similar to CF, DPB P. aeruginosa strains produce a diverse array of modified lipid A structures. Antibiotic susceptibility testing revealed increased resistance to erythromycin, a representative agent of the macrolide antibiotics used to manage DPB patients. Differences in the O-antigen type among P. aeruginosa strains collected from these different backgrounds were also observed. Ultimately, the characterization of DPB P. aeruginosa strains highlights several unique qualities of P. aeruginosa strains collected from chronically diseased airways, underscoring the challenges in treating DPB, CF, and other obstructive respiratory disease patients with P. aeruginosa infections.IMPORTANCEDiffuse panbronchiolitis (DPB), a chronic lung disease characterized by persistent P. aeruginosa infection, serves as an informative comparator to more common chronic lung diseases, such as cystic fibrosis (CF). This study aimed to better address the interplay between P. aeruginosa and chronically compromised airway environments through the examination of DPB P. aeruginosa strains, as existing literature regarding DPB is limited to case reports, case series, and clinical treatment guidelines. The evaluation of these features in the context of DPB, in tandem with prevailing knowledge of P. aeruginosa strains collected from more common chronic lung diseases (e.g., CF), can aid in the development of more effective strategies to combat respiratory P. aeruginosa infections in patients with chronic lung diseases.

BackgroundThis study unveils the intricate functional association between cyclic di-3’,5’-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive.MethodsWe employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn.ResultsWe demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile.ConclusionThe multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

ABSTRACT Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from the host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that Rickettsia akari (TRG), Rickettsia typhi (TG), and Rickettsia montanensis (SFG) produce lipid A with long 2′ secondary acyl chains (C16 or C18) compared to short 2′ secondary acyl chains (C12) in Rickettsia rickettsii (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2′ secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae (Rickettsia rhipicephali and Rickettsia parkeri) utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLATn-derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2′ secondary acyl chains. While 2′ secondary acyl chain lengths do not distinguish Rickettsia pathogens from non-pathogens, in silico analyses of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2′ secondary acyl chain addition. Our collective data warrant determining Rickettsia lipid A inflammatory potential and how structural heterogeneity impacts lipid A-host receptor interactions.IMPORTANCEDeforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in Rickettsia rickettsii (later-evolving SFG) relative to Rickettsia montanensis (basal SFG), Rickettsia typhi (TG), and Rickettsia akari (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of Rickettsia lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of ex vivo lipid A chemical extraction by utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry, a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm that later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different Rickettsia pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.

ABSTRACT Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2′-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2′-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal–oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.

ABSTRACT Spatially aware de novo discovery methods are essential tools for therapeutic target identification in complex interphylum interactions such as arthropods and mammals. Notably, the methods should ideally be species agnostic, showing unique features of all interacting species. We evaluated the possibilities for matrix-assisted desorption/ionization mass spectrometry imaging (MALDI-MSI, referred to here as MSI) as a spatial “omics” method to simultaneously profile both an arthropod vector (Ixodes tick) and a mammalian skin (guinea pig) in a bite model. We demonstrated the feasibility of MSI using gelatin-stabilized sample mounting that allowed for serial sectioning and mapping lipids in control and bitten skin, including the tick body and embedded mouthparts. We identified unique lipid ion patterns and observed lipid reorganization beneath the bite site consistent with histological changes. Furthermore, several ions were observed in the tick body with lower intensity in the dermis and control skin, suggesting the transmission of lipids from the tick to mammalian skin. These results establish a multi-system approach for discovering cross-species molecular interactions that can be further developed as targets to disrupt the vector-host interface.IMPORTANCEHere, we demonstrate the adaptability of spatial “omics” methods to identify interphylum processes regulated at the vector-host interface of ticks during a mammalian blood meal. This approach enables a better understanding of complex bipartite or tripartite molecular interactions between hosts, arthropod vectors and transmitted pathogens, and contributes toward the development of spatially aware therapeutic target discovery and description.

Abstract The opportunistic pathogen Pseudomonas aeruginosa (Pa) causes severe nosocomial infections, especially in immunocompromised individuals and the elderly. Increasing drug resistance, the absence of a licensed vaccine and increased hospitalizations due to SARS-CoV-2 have made Pa a major healthcare risk. To address this, we formulated a candidate subunit vaccine against Pa (L-PaF), by fusing the type III secretion system tip and translocator proteins with LTA1 in an oil-in-water emulsion (ME). This was mixed with the TLR4 agonist (BECC438b). Lung mRNA sequencing showed that the formulation activates genes from multiple immunological pathways eliciting a protective Th1-Th17 response following IN immunization. Following infection, however, the immunized mice showed an adaptive response while the PBS-vaccinated mice experienced rapid onset of an inflammatory response. The latter displayed a hypoxic lung environment with high bacterial burden. Finally, the importance of IL-17 and immunoglobulins were demonstrated using knockout mice. These findings suggest a need for a balanced humoral and cellular response to prevent the onset of Pa infection and that our formulation could elicit such a response.

Abstract Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Abstract Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA + BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~ 12-month-old mouse aged model. Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470.

Longitudinal proteomics holds great promise for biomarker discovery, but the data interpretation has remained a challenge. Here, the authors evaluate several tools to detect longitudinal differential expression in proteomics data and introduce RolDE, a robust reproducibility optimization approach.

Abstract Whole cell vaccines are complex mixtures of antigens, immunogens, and sometimes adjuvants that can trigger potent and protective immune responses. In some instances, such as whole cell Bordetella pertussis vaccination, the immune response to vaccination extends beyond the pathogen the vaccine was intended for and contributes to protection against other clinically significant pathogens. In this study, we describe how B. pertussis whole cell vaccination protects mice against acute pneumonia caused by Pseudomonas aeruginosa. Using ELISA and western blot, we identified that B. pertussis whole cell vaccination induces production of antibodies that bind to lab-adapted and clinical strains of P. aeruginosa, regardless of immunization route or adjuvant used. The cross-reactive antigens were identified using immunoprecipitation, mass spectrometry, and subsequent immunoblotting. We determined that B. pertussis GroEL and OmpA present in the B. pertussis whole cell vaccine led to production of antibodies against P. aeruginosa GroEL and OprF, respectively. Finally, we showed that recombinant B. pertussis OmpA was sufficient to induce protection against P. aeruginosa acute murine pneumonia. This study highlights the potential for use of B. pertussis OmpA as a vaccine antigen for prevention of P. aeruginosa infection, and the potential of broadly protective antigens for vaccine development.

Shigellosis (bacillary dysentery) is a severe gastrointestinal infection with a global incidence of 90 million cases annually. Despite the severity of this disease, there is currently no licensed vaccine against shigellosis. Shigella’s primary virulence factor is its type III secretion system (T3SS), which is a specialized nanomachine used to manipulate host cells. A fusion of T3SS injectisome needle tip protein IpaD and translocator protein IpaB, termed DBF, when admixed with the mucosal adjuvant double-mutant labile toxin (dmLT) from enterotoxigenic E. coli was protective using a murine pulmonary model. To facilitate the production of this platform, a recombinant protein that consisted of LTA-1, the active moiety of dmLT, and DBF were genetically fused, resulting in L-DBF, which showed improved protection against Shigella challenge. To extrapolate this protection from mice to humans, we modified the formulation to provide for a multivalent presentation with the addition of an adjuvant approved for use in human vaccines. Here, we show that L-DBF formulated (admix) with a newly developed TLR4 agonist called BECC438 (a detoxified lipid A analog identified as Bacterial Enzymatic Combinatorial Chemistry candidate #438), formulated as an oil-in-water emulsion, has a very high protective efficacy at low antigen doses against lethal Shigella challenge in our mouse model. Optimal protection was observed when this formulation was introduced at a mucosal site (intranasally). When the formulation was then evaluated for the immune response it elicits, protection appeared to correlate with high IFN-γ and IL-17 secretion from mucosal site lymphocytes.

Summary: Pseudomonas aeruginosa (Pa) is a pathogen causing chronic pulmonary infections in patients with cystic fibrosis (CF). Manipulation of lipids is an important feature of Pa infection and on a tissue-level scale is poorly understood. Using a mouse model of acute Pa pulmonary infection, we explored the whole-lung phospholipid response using mass spectrometry imaging (MSI) and spatial lipidomics. Using a histology-driven analysis, we isolated airways and parenchyma from both mock- and Pa-infected lungs and used systems biology tools to identify enriched metabolic pathways from the differential phospholipid identities. Infection was associated with a set of 26 ions, with 11 unique to parenchyma and 6 unique to airways. Acyl remodeling was differentially enriched in infected parenchyma as the predominant biological function. These functions correlated with markers of polymorphonuclear (PMN) cell influx, a defining feature of the lung response to Pa infection, implicating enzymes active in phospholipid remodeling.

ABSTRACT Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient’s lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4′ phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules’ biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

A longitudinal study in mice reveals that a booster dose of mRNA vaccine BNT162b2 enhances humoral and cell-mediated responses and provides sterilizing immunity against Omicron-induced lung infection in aged animals.

Salmonella enterica, a Gram-negative pathogen, has over 2500 serovars that infect a wide range of hosts. In humans, S. enterica causes typhoid or gastroenteritis and is a major public health concern. In this study, SseB (the tip protein of the Salmonella pathogenicity island 2 type III secretion system) was fused with the LTA1 subunit of labile-toxin from enterotoxigenic E. coli to make the self-adjuvanting antigen L-SseB. Two unique nanoparticle formulations were developed to allow multimeric presentation of L-SseB. Mice were vaccinated with these formulations and protective efficacy determined via challenging the mice with S. enterica serovars. The polysaccharide (chitosan) formulation was found to elicit better protection when compared to the squalene nanoemulsion. When the polysaccharide formulation was used to vaccinate rabbits, protection from S. enterica challenge was elicited. In summary, L-SseB in a particulate polysaccharide formulation appears to be an attractive candidate vaccine capable of broad protection against S. enterica.

Lipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the Leptospira genus was explored. The individual Leptospira species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 Leptospira reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of Leptospira membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on Leptospira lipid A activity.

Abstract SARS-CoV-2 is a viral respiratory pathogen responsible for the current global pandemic and the disease that causes COVID-19. All current WHO approved COVID-19 vaccines are administered through the muscular route. We have developed a prototype two-dose vaccine (BReC-CoV-2) by combining the Receptor Binding Domain (RBD) antigen, via conjugation to Diphtheria toxoid (EcoCRM®). The vaccine is adjuvanted with Bacterial Enzymatic Combinatorial Chemistry (BECC), BECC470. Intranasal (IN) administration of BreC-CoV-2 in K18-hACE2 mice induced a strong systemic and localized immune response in the respiratory tissues which provided protection against the Washington strain of SARS-CoV-2. Protection provided after IN administration of BReC-CoV-2 was associated with decreased viral RNA copies in the lung, robust RBD IgA titers in the lung and nasal wash, and induction of broadly neutralizing antibodies in the serum. We also observed that BReC-CoV-2 vaccination administered using an intramuscular (IM) prime and IN boost protected mice from a lethal challenge dose of the Delta variant of SARS-CoV-2. IN administration of BReC-CoV-2 provided better protection than IM only administration to mice against lethal challenge dose of SARS-CoV-2. These data suggest that the IN route of vaccination induces localized immune responses that can better protect against SARS-CoV-2 than the IM route in the upper respiratory tract.

ABSTRACT Urinary tract infections (UTIs) pose a major public health burden. The vast majority of UTIs are caused by Gram-negative bacteria. Current culture-based pathogen identification methods may require up to 24 to 48 h of incubation. In this study, we developed and evaluated a method for Gram-negative pathogen identification direct from urine, without culture, via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in approximately 1 h. Urine samples were collected (n = 137) from the University of Maryland Medical Center clinical microbiology laboratory. To identify bacteria direct from urine, two methods were evaluated. First, 1 μL of urine was directly spotted onto the MALDI target plate, and second, 1 mL of urine was centrifuged at 8,000 rpm for 5 min before processing using the fast lipid analysis technique (FLAT). Mass spectra were acquired on the Bruker MALDI Biotyper sirius system in the negative-ion mode. Results were compared to those of standard culture methods. When 1 μL of urine was directly spotted, positive agreement was 81.5% (101/124) and, after centrifugation, 94.4% (117/124) relative to that of standard culture methods. Negative agreement for both methods was 100% (13/13). The time to results for both of the specimen preparation methods using the FLAT extraction protocol was approximately 1 h, with minimal hands-on time required (

ABSTRACT Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.

Summary: Pseudomonas aeruginosa infections can be difficult to treat and new therapeutics are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and used them to screen and isolate over a dozen P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. We evolved bacterial mutants that were resistant to phage infection for four different phages, and used genome sequencing and functional analysis to study them further. We also tested phages for their ability to kill P. aeruginosa grown in biofilms in vitro and ex vivo on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

ABSTRACT Due to its high transmissibility, Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Here, we studied the biological cost of colistin resistance, an antibiotic of last resort, in this opportunistic pathogen using a murine model of gut colonization and transmission. Colistin resistance in K. pneumoniae is commonly the result of the inactivation of the small regulatory protein MgrB. Without a functional MgrB, the two-component system PhoPQ is constitutively active, leading to an increase in lipid A modifications and subsequent colistin resistance. Using an isogenic mgrB deletion mutant (MgrB−), we demonstrate that the mutant’s colistin resistance is not associated with a fitness defect under in vitro growth conditions. However, in our murine model of K. pneumoniae gastrointestinal (GI) colonization, the MgrB− colonizes the gut poorly, allowing us to identify a fitness cost. Moreover, the MgrB− mutant has higher survival outside the host compared with the parental strain. We attribute this enhanced survivability to dysregulation of the PhoPQ two-component system and accumulation of the master stress regulator RpoS. The enhanced survival of MgrB− may be critical for its rapid host-to-host transmission observed in our model. Together, our data using multiple clinical isolates demonstrate that MgrB-dependent colistin resistance in K. pneumoniae comes with a biological cost in gut colonization. However, this cost is mitigated by enhanced survival outside the host and consequently increases its host-to-host transmission. Additionally, it underscores the importance of considering the entire life cycle of a pathogen to determine the actual biological cost associated with antibiotic resistance. IMPORTANCE The biological cost associated with colistin resistance in Klebsiella pneumoniae was examined using a murine model of K. pneumoniae gut colonization and fecal-oral transmission. A common mutation resulting in colistin resistance in K. pneumoniae is a loss-of-function mutation of the small regulatory protein MgrB that regulates the two-component system PhoPQ. Even though colistin resistance in K. pneumoniae comes with a fitness defect in gut colonization, it increases bacterial survival outside the host enabling it to transmit more effectively to a new host. The enhanced survival is dependent upon the accumulation of RpoS and dysregulation of the PhoPQ. Hence, our study expands our understanding of the underlying molecular mechanism contributing to the transmission of colistin-resistant K. pneumoniae.

Abstract Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.

ABSTRACT Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill50) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient’s infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections in humans. In addition to its innate antibiotic resistance, P. aeruginosa is very effective in acquiring resistance resulting in the emergence of multi-drug resistance strains and a licensed vaccine is not yet available. We have previously demonstrated the protective efficacy of a novel antigen PaF (Pa Fusion), a fusion of the type III secretion system (T3SS) needle tip protein, PcrV, and the first of two translocator proteins, PopB. PaF was modified to provide a self-adjuvanting activity by fusing the A1 subunit of the heat-labile enterotoxin from Enterotoxigenic E. coli to its N-terminus to give L-PaF. In addition to providing protection against 04 and 06 serotypes of P. aeruginosa, L-PaF elicited opsonophagocytic killing and stimulated IL-17A secretion, which have been predicted to be required for a successful vaccine. While monomeric recombinant subunit vaccines can be protective in mice, this protection often does not transfer to humans where multimeric formulations perform better. Here, we use two unique formulations, an oil-in-water (o/w) emulsion and a chitosan particle, as well as the addition of a unique TLR4 agonist, BECC438 (a detoxified lipid A analogue designated Bacterial Enzymatic Combinatorial Chemistry 438), as an initial step in optimizing L-PaF for use in humans. The o/w emulsion together with BECC438 provided the best protective efficacy, which correlated with high levels of opsonophagocytic killing and IL-17A secretion, thereby reducing the lung burden among all the vaccinated groups tested.

Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial innate immune response to bacterial infection and the tissue damage. This response is facilitated by specific receptors on the cell surface and intracellularly.

Abstract Mass spectrometry imaging (MSI) is a technique for mapping the spatial distributions of molecules in sectioned tissue. Histology-preserving tissue preparation methods are central to successful MSI studies. Common fixation methods, used to preserve tissue morphology, can result in artifacts in the resulting MSI experiment including delocalization of analytes, altered adduct profiles, and loss of key analytes due to irreversible cross-linking and diffusion. This is especially troublesome in lung and airway samples, in which histology and morphology is best interpreted from 3D reconstruction, requiring the large and small airways to remain inflated during analysis. Here, we developed an MSI-compatible inflation containing as few exogenous components as possible, forgoing perfusion, fixation, and addition of salt solutions upon inflation that resulted in an ungapped 3D molecular reconstruction through more than 300 microns. We characterized a series of polyunsaturated phospholipids (PUFA-PLs), specifically phosphatidylinositol (-PI) lipids linked to lethal inflammation in bacterial infection and mapped them in serial sections of inflated mouse lung. PUFA-PIs were identified using spatial lipidomics and determined to be determinant markers of major airway features using unsupervised hierarchical clustering. Deep lung architecture was preserved using this inflation approach and the resulting sections are compatible with multiple MSI modalities, automated interpretation software, and serial 3D reconstruction.

Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia

Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy.

Abstract Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa, and E nterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.

The insect IMD signalling pathway detects invading pathogens. Here the authors show that ticks have an alternative IMD system that lacks peptidoglycan receptors, IMD and FADD, and is instead reliant on interaction of the E3 ligase XIAP with the E2 conjugating enzyme Bendless.

ABSTRACT The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens. IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem- and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant “superbugs.”

The innate immune system serves as a first line of defense against microbial pathogens. The host innate immune response can be triggered by recognition of conserved non-self-microbial signature molecules by specific host receptor proteins called Toll-like receptors. For bacteria, many of these molecular triggers reside on or are embedded in the bacterial membrane, the interface exposed to the host environment. Lipids are the most abundant component of membranes, and bacteria possess a unique set of lipids that can initiate or modify the host innate immune response. Bacterial lipoproteins, peptidoglycan, and outer membrane molecules lipoteichoic acid and lipopolysaccharide are key modulators of the host immune system. This review article will highlight some of the research emerging at the crossroads of bacterial membranes and innate immunity.

ABSTRACT Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates. IMPORTANCE There is an urgent need to develop effective vaccines against infectious diseases that continue to be major causes of morbidity and mortality worldwide. Making effective vaccines requires selecting an adjuvant to strengthen an appropriate and protective immune response. This work describes a practical method, bacterial enzymatic combinatorial chemistry (BECC), for generating functionally diverse molecules for adjuvant use. These molecules were analyzed in cell culture for their ability to initiate immune stimulatory activity. Several of the assays described herein show promising in vitro cytokine production and costimulatory molecule expression results, suggesting that the BECC molecules may be useful in future vaccine preparations.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.

ABSTRACT To gain a better understanding of the genes and proteins involved in group A Streptococcus (GAS; Streptococcus pyogenes) biofilm growth, we analyzed the transcriptome, cellular proteome, and cell wall proteome from biofilms at different stages and compared them to those of plankton-stage GAS. Using high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we found distinct expression profiles in the transcriptome and proteome. A total of 46 genes and 41 proteins showed expression across the majority of biofilm time points that was consistently higher or consistently lower than that seen across the majority of planktonic time points. However, there was little overlap between the genes and proteins on these two lists. In line with other studies comparing transcriptomic and proteomic data, the overall correlation between the two data sets was modest. Furthermore, correlation was poorest for biofilm samples. This suggests a high degree of regulation of protein expression by nontranscriptional mechanisms. This report illustrates the benefits and weaknesses of two different approaches to global expression profiling, and it also demonstrates the advantage of using proteomics in conjunction with transcriptomics to gain a more complete picture of global expression within biofilms. In addition, this report provides the fullest characterization of expression patterns in GAS biofilms currently available. IMPORTANCE Prokaryotes are thought to regulate their proteomes largely at the level of transcription. However, the results from this first set of global transcriptomic and proteomic analyses of paired microbial samples presented here show that this assumption is false for the majority of genes and their products in S. pyogenes. In addition, the tenuousness of the link between transcription and translation becomes even more pronounced when microbes exist in a biofilm or a stationary planktonic state. Since the transcriptome level does not usually equal the proteome level, the validity attributed to gene expression studies as well as proteomic studies in microbial analyses must be brought into question. Therefore, the results attained by either approach, whether RNA-seq or shotgun proteomics, must be taken in context and evaluated with particular care since they are by no means interchangeable.