High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

Staphylococcus aureus Overcomes Anaerobe-Derived Short-Chain Fatty Acid Stress via FadX and the CodY Regulon.

Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coenzyme A (CoA)-transferase, was significantly increased, leading to our hypothesis that short-chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was observed frequently. Whole-genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its cocolonizing microbiota and metabolites they exchange and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth. IMPORTANCE Staphylococcus aureus is an important CRS pathogen, and yet it is found in the upper airways of 30% to 50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, namely, Fusobacterium nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota.

Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.

In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

Human fecal metabolomic profiling could inform Clostridioides difficile infection diagnosis and treatment.

Clostridioides difficile is a significant public health threat, and diagnosis of this infection is challenging due to a lack of sensitivity in current diagnostic testing. In this issue of the JCI, Robinson et al. use a logistic regression model based on the fecal metabolome that is able to distinguish between patients with non-C. difficile diarrhea and C. difficile infection, and to some degree, patients who are asymptomatically colonized with C. difficile. The authors construct a metabolic definition of human C. difficile infection, which could improve diagnostic accuracy and aid in the development of targeted therapeutics against this pathogen.

The Ability to Acquire Iron Is Inversely Related to Virulence and the Protective Efficacy of Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a highly infectious bacterial pathogen that causes the potentially fatal disease tularemia. The Live Vaccine Strain (LVS) of F. tularensis subsp. holarctica, while no longer licensed as a vaccine, is used as a model organism for identifying correlates of immunity and bacterial factors that mediate a productive immune response against F. tularensis. Recently, it was reported that two biovars of LVS differed in their virulence and vaccine efficacy. Genetic analysis showed that they differ in ferrous iron homeostasis; lower Fe2+ levels contributed to increased resistance to hydrogen peroxide in the vaccine efficacious LVS biovar. This also correlated with resistance to the bactericidal activity of interferon γ-stimulated murine bone marrow-derived macrophages. We have extended these findings further by showing that a mutant lacking bacterioferritin stimulates poor protection against Schu S4 challenge in a mouse model of tularemia. Together these results suggest that the efficacious biovar of LVS stimulates productive immunity by a mechanism that is dependent on its ability to limit the toxic effects of oxidative stress by maintaining optimally low levels of intracellular Fe2+.

Shifts in the Gut Metabolome and Clostridium difficile Transcriptome throughout Colonization and Infection in a Mouse Model.

Antibiotics alter the gut microbiota and decrease resistance to Clostridium difficile colonization; however, the mechanisms driving colonization resistance are not well understood. Loss of resistance to C. difficile colonization due to antibiotic treatment is associated with alterations in the gut metabolome, specifically, with increases in levels of nutrients that C. difficile can utilize for growth in vitro. To define the nutrients that C. difficile requires for colonization and pathogenesis in vivo, we used a combination of mass spectrometry and RNA sequencing (RNA Seq) to model the gut metabolome and C. difficile transcriptome throughout an acute infection in a mouse model at the following time points: 0, 12, 24, and 30 h. We also performed multivariate-based integration of the omics data to define the signatures that were most important throughout colonization and infection. Here we show that amino acids, in particular, proline and branched-chain amino acids, and carbohydrates decrease in abundance over time in the mouse cecum and that C. difficile gene expression is consistent with their utilization in vivo. This was also reinforced by the multivariate-based integration of the omics data where we were able to discriminate the metabolites and transcripts that support C. difficile physiology between the different time points throughout colonization and infection. This report illustrates how important the availability of amino acids and other nutrients is for the initial stages of C. difficile colonization and progression of disease. Future studies identifying the source of the nutrients and engineering bacteria capable of outcompeting C. difficile in the gut will be important for developing new targeted bacterial therapeutics. IMPORTANCE Clostridium difficile is a bacterial pathogen of global significance that is a major cause of antibiotic-associated diarrhea. Antibiotics deplete the indigenous gut microbiota and change the metabolic environment in the gut to one favoring C. difficile growth. Here we used metabolomics and transcriptomics to define the gut environment after antibiotics and during the initial stages of C. difficile colonization and infection. We show that amino acids, in particular, proline and branched-chain amino acids, and carbohydrates decrease in abundance over time and that C. difficile gene expression is consistent with their utilization by the bacterium in vivo. We employed an integrated approach to analyze the metabolome and transcriptome to identify associations between metabolites and transcripts. This highlighted the importance of key nutrients in the early stages of colonization, and the data provide a rationale for the development of therapies based on the use of bacteria that specifically compete for nutrients that are essential for C. difficile colonization and disease.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis.

T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis spp. tularensis (Ftt). The general paucity of novel vaccines for Ftt during the past 60 y can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus epitopes, we found that survival correlated with persistence of Ag-specific CD4(+) T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the Ag-specific response was underscored by our observation that inclusion of an epitope that elicits high-avidity CD4(+) T cells converted a poorly protective vaccine to one that engenders 100% protection. Taken together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate Ag-specific CD4(+) T cell responses and that elucidation of Francisella epitopes that elicit high-avidity CD4(+) T cell responses, specifically in humans, will be required for successful vaccine development.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.

Characterization of Francisella tularensis Schu S4 mutants identified from a transposon library screened for O-antigen and capsule deficiencies.

The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen.

Uncovering the components of the Francisella tularensis virulence stealth strategy.

Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies.

The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp.

The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

Epigenetic regulation of the calcitonin gene-related peptide gene in trigeminal glia.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. METHODS: DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. RESULTS: A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. CONCLUSIONS: Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.