Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

Critical role of growth medium for detecting drug interactions in Gram-negative bacteria that model in vivo responses.

The rise in infections caused by multidrug-resistant (MDR) bacteria has necessitated a variety of clinical approaches, including the use of antibiotic combinations. Here, we tested the hypothesis that drug-drug interactions vary in different media, and determined which in vitro models best predict drug interactions in the lungs. We systematically studied pair-wise antibiotic interactions in three different media, CAMHB, (a rich lab medium standard for antibiotic susceptibility testing), a urine mimetic medium (UMM), and a minimal medium of M9 salts supplemented with glucose and iron (M9Glu) with three Gram-negative ESKAPE pathogens, Acinetobacter baumannii (Ab), Klebsiella pneumoniae (Kp), and Pseudomonas aeruginosa (Pa). There were pronounced differences in responses to antibiotic combinations between the three bacterial species grown in the same medium. However, within species, PaO1 responded to drug combinations similarly when grown in all three different media, whereas Ab17978 and other Ab clinical isolates responded similarly when grown in CAMHB and M9Glu medium. By contrast, drug interactions in Kp43816, and other Kp clinical isolates poorly correlated across different media. To assess whether any of these media were predictive of antibiotic interactions against Kp in the lungs of mice, we tested three antibiotic combination pairs. In vitro measurements in M9Glu, but not rich medium or UMM, predicted in vivo outcomes. This work demonstrates that antibiotic interactions are highly variable across three Gram-negative pathogens and highlights the importance of growth medium by showing a superior correlation between in vitro interactions in a minimal growth medium and in vivo outcomes. IMPORTANCE: Drug-resistant bacterial infections are a growing concern and have only continued to increase during the SARS-CoV-2 pandemic. Though not routinely used for Gram-negative bacteria, drug combinations are sometimes used for serious infections and may become more widely used as the prevalence of extremely drug-resistant organisms increases. To date, reliable methods are not available for identifying beneficial drug combinations for a particular infection. Our study shows variability across strains in how drug interactions are impacted by growth conditions. It also demonstrates that testing drug combinations in tissue-relevant growth conditions for some strains better models what happens during infection and may better inform combination therapy selection.

Role of the Yersinia pseudotuberculosis Virulence Plasmid in Pathogen-Phagocyte Interactions in Mesenteric Lymph Nodes.

Yersinia pseudotuberculosis is an Enterobacteriaceae family member that is commonly transmitted by the fecal-oral route to cause infections. From the small intestine, Y. pseudotuberculosis can invade through Peyer's patches and lymph vessels to infect the mesenteric lymph nodes (MLNs). Infection of MLNs by Y. pseudotuberculosis results in the clinical presentation of mesenteric lymphadenitis. MLNs are important for immune responses to intestinal pathogens and microbiota in addition to their clinical relevance to Y. pseudotuberculosis infections. A characteristic of Y. pseudotuberculosis infection in MLNs is the formation of pyogranulomas. Pyogranulomas are composed of neutrophils, inflammatory monocytes, and lymphocytes surrounding extracellular microcolonies of Y. pseudotuberculosis. Key elements of the complex pathogen-host interaction in MLNs have been identified using mouse infection models. Y. pseudotuberculosis requires the virulence plasmid pYV to induce the formation of pyogranulomas in MLNs. The YadA adhesin and the Ysc-Yop type III secretion system (T3SS) are encoded on pYV. YadA mediates bacterial binding to host receptors, which engages the T3SS to preferentially translocate seven Yop effectors into phagocytes. The effectors promote pathogenesis by blocking innate immune defenses such as superoxide production, degranulation, and inflammasome activation, resulting in survival and growth of Y. pseudotuberculosis. On the other hand, certain effectors can trigger immune defenses in phagocytes. For example, YopJ triggers activation of caspase-8 and an apoptotic cell death response in monocytes within pyogranulomas that limits dissemination of Y. pseudotuberculosis from MLNs to the bloodstream. YopE can be processed as an antigen by phagocytes in MLNs, resulting in T and B cell responses to Y. pseudotuberculosis. Immune responses to Y. pseudotuberculosis in MLNs can also be detrimental to the host in the form of chronic lymphadenopathy. This review focuses on interactions between Y. pseudotuberculosis and phagocytes mediated by pYV that concurrently promote pathogenesis and host defense in MLNs. We propose that MLN pyogranulomas are immunological arenas in which opposing pYV-driven forces determine the outcome of infection in favor of the pathogen or host.

Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers.

Many pathogens use M cells to access the underlying Peyer's patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb), and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyer's patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.

Signaling cascades converting the recognition of pathogens to efficient inflammatory responses by neutrophils are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following neutrophil stimulation by integrins, formyl peptide receptors, and for host defense against the Gram-negative bacterial pathogens, Klebsiella pneumoniae and Yersinia pseudotuberculosis. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 in neutrophils is crucial for maximal ROS response to purified C-type lectin receptor agonists and to the fungal pathogens, Candida glabrata and Candida albicans, and for robust killing of C. glabrata. Inside-out signaling to integrin and Syk phosphorylation occurred independently of SKAP2 after Candida infection. However, Pyk2, ERK1/2, and p38 phosphorylation were significantly reduced after infection with C. glabrata and K. pneumoniae in Skap2-/- neutrophils. These data demonstrate the importance of SKAP2 in ROS generation and host defense beyond antibacterial immunity to include CLRs and Candida species.

A Structure-Function-Inhibition Analysis of the Pseudomonas aeruginosa Type III Secretion Needle Protein PscF.

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

SKAP2 is required for defense against K. pneumoniae infection and neutrophil respiratory burst.

Klebsiella pneumoniae is a respiratory, blood, liver, and bladder pathogen of significant clinical concern. We show that the adaptor protein, SKAP2, is required for protection against K. pneumoniae (ATCC 43816) pulmonary infections. Skap2-/- mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells. Skap2-/- neutrophils and monocytes were present in infected lungs, and the neutrophils degranulated normally in response to K. pneumoniae infection in mice; however, K. pneumoniae-stimulated reactive oxygen species (ROS) production in vitro was abolished. K. pneumoniae-induced neutrophil ROS response required the activity of SFKs, Syk, Btk, PLCγ2, and PKC. The loss of SKAP2 significantly hindered the K. pneumoniae-induced phosphorylation of SFKs, Syk, and Pyk2 implicating SKAP2 as proximal to their activation in pathogen-signaling pathways. In conclusion, SKAP2-dependent signaling in neutrophils is essential for K. pneumoniae-activated ROS production and for promoting bacterial clearance during infection.

Klebsiella pneumoniae is a type of bacteria that can cause life-threatening infections ­ including pneumonia, blood stream infections, and urinary tract infections ­ in hospitalized patients. These infections can be difficult to treat because some K. pneumoniae are resistant to antibiotics. The bacteria are normally found in the human intestine, and they do not usually cause infections in healthy people. This implies that healthy people's immune systems are better able to fend off K. pneumoniae infections; learning how could help scientists develop new ways to treat or prevent infections in hospitalized patients. In healthy people, a type of immune cell called neutrophils are the first line of defense against bacterial infections. Several different proteins are needed to activate neutrophils, including a protein called SKAP2. But the role of this protein in fighting K. pneumoniae infections is not clear. To find out what role SKAP2 plays in the defense against pneumonia caused by K. pneumoniae, Nguyen et al. compared infections in mice with and without the protein. Mice lacking SKAP2 in their white blood cells had more bacteria in their lungs than normal mice. The experiments showed that neutrophils from mice with SKAP2 produce a burst of chemicals called "reactive oxygen species", which can kill bacteria. But neutrophils without the protein do not. Without SKAP2, several proteins that help produce reactive oxygen species do not work. Understanding the role of SKAP2 in fighting infections may help scientists better understand the immune system. This could help clinicians to treat conditions that cause it to be hyperactive or ineffective. More studies are needed to determine if SKAP2 works the same way in human neutrophils and if it works against all types of K. pneumoniae. If it does, then scientists might be able use this information to develop therapies that help the immune system fight infections.

Yersinia pseudotuberculosis YopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.

Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice.

Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer's patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-α to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

Unraveling neutrophil- Yersinia interactions during tissue infection.

The human and animal pathogens Yersinia pestis, which causes bubonic and pneumonic plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica, which cause gastroenteritis, share a type 3 secretion system which injects effector proteins, Yops, into host cells. This system is critical for virulence of all three pathogens in tissue infection. Neutrophils are rapidly recruited to infected sites and all three pathogens frequently interact with and inject Yops into these cells during tissue infection. Host receptors, serum factors, and bacterial adhesins appear to collaborate to promote neutrophil- Yersinia interactions in tissues. The ability of neutrophils to control infection is mixed depending on the stage of infection and points to the efficiency of Yops and other bacterial factors to mitigate bactericidal effects of neutrophils. Yersinia in close proximity to neutrophils has higher levels of expression from yop promoters, and neutrophils in close proximity to Yersinia express higher levels of pro-survival genes than migrating neutrophils. In infected tissues, YopM increases neutrophil survival and YopH targets a SKAP2/SLP-76 signal transduction pathway. Yet the full impact of these and other Yops and other Yersinia factors on neutrophils in infected tissues has yet to be understood.

Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using ß-Lactamase Fusions.

Development of the TEM-CCF2/4-AM FRET-based system has enabled investigators to track translocation of effector proteins into mammalian cells during infection. This allows for separation of translocated and non-translocated cell populations for further study. Yersinia strains expressing translational Yop-TEM fusions, containing the secretion and translocation signals of a Yop with the TEM-1 portion of ß-lactamase, are used to infect mice, tissues isolated from mice, or mammalian cells in culture. Infected and harvested mammalian cells are treated with either CCF2-AM or CCF4-AM, and cleavage of this fluorescent compound by TEM is detected by fluorescence-activated cell sorting (FACS) analysis. A shift from green to blue emission spectra of individual cells is indicative of translocation of a given Yop-TEM fusion protein into the host cell during Yersinia infection due to a disruption in FRET between the two fluors of the compound. In Yersinia, this method has been used to understand Type III secretion dynamics and Yop functions in cells translocated by effectors during infection. Here, we describe how to generate Yop-TEM constructs, and how to detect, quantify, isolate, and study Yop-TEM containing cells in murine tissues during infection and in ex vivo tissues by cell sorting and flow cytometry analysis. In addition, we provide guidance for analyzing TEM-positive cells via a plate reader and fluorescent microscopy.

Amino Acid Biosynthetic Pathways Are Required for Klebsiella pneumoniae Growth in Immunocompromised Lungs and Are Druggable Targets during Infection.

The emergence of multidrug-resistant Klebsiella pneumoniae has rendered a large array of infections difficult to treat. In a high-throughput genetic screen of factors required for K. pneumoniae survival in the lung, amino acid biosynthesis genes were critical for infection in both immunosuppressed and wild-type (WT) mice. The limited pool of amino acids in the lung did not change during infection and was insufficient for K. pneumoniae to overcome attenuating mutations in aroA, hisA, leuA, leuB, serA, serB, trpE, and tyrA in WT and immunosuppressed mice. Deletion of aroA, which encodes 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase class I, resulted in the most severe attenuation. Treatment with the EPSP synthase-specific competitive inhibitor glyphosate decreased K. pneumoniae growth in the lungs. K. pneumoniae expressing two previously identified glyphosate-resistant mutations in EPSP synthase had significant colonization defects in lung infection. Selection and characterization of six spontaneously glyphosate-resistant mutants in K. pneumoniae yielded no mutations in aroA Strikingly, glyphosate treatment of mice lowered the bacterial burden of two of three spontaneous glyphosate-resistant mutants and further lowered the burden of the less-attenuated EPSP synthase catalytic mutant. Of 39 clinical isolate strains, 9 were resistant to glyphosate at levels comparable to those of selected resistant strains, and none appeared to be more highly resistant. These findings demonstrate amino acid biosynthetic pathways essential for K. pneumoniae infection are promising novel therapeutic targets.

New Age Strategies To Reconstruct Mucosal Tissue Colonization and Growth in Cell Culture Systems.

Over the past few decades, in vitro cell culture systems have greatly expanded our understanding of host-pathogen interactions. However, studies using these models have been limited by the fact that they lack the complexity of the human body. Therefore, recent efforts that allow tissue architecture to be mimicked during in vitro culture have included the development of methods and technology that incorporate tissue structure, cellular composition, and efficient long-term culture. These advances have opened the door for the study of pathogens that previously could not be cultured and for the study of pathophysiological properties of infection that could not be easily elucidated using traditional culture models. Here we discuss the latest studies using organoids and engineering technology that have been developed and applied to the study of host-pathogen interactions in mucosal tissues.

Promises and Challenges of the Type Three Secretion System Injectisome as an Antivirulence Target.

Antibiotic resistance is a major public health threat that has stimulated the scientific community to search for nontraditional therapeutic targets. Because virulence, but not the growth, of many Gram-negative bacterial pathogens depends on the multicomponent type three secretion system injectisome (T3SSi), the T3SSi has been an attractive target for identifying small molecules, peptides, and monoclonal antibodies that inhibit its function to render the pathogen avirulent. While many small-molecule lead compounds have been identified in whole-cell-based high-throughput screens (HTSs), only a few protein targets of these compounds are known; such knowledge is an important step to developing more potent and specific inhibitors. Evaluation of the efficacy of compounds in animal studies is ongoing. Some efforts involving the development of antibodies and vaccines that target the T3SSi are further along and include an antibody that is currently in phase II clinical trials. Continued research into these antivirulence therapies, used alone or in combination with traditional antibiotics, requires combined efforts from both pharmaceutical companies and academic labs.

Pre-colonization with the commensal fungus Candida albicans reduces murine susceptibility to Clostridium difficile infection.

Clostridium difficile is a major nosocomial pathogen responsible for close to half a million infections and 27,000 deaths annually in the U.S. Preceding antibiotic treatment is a major risk factor for C. difficile infection (CDI) leading to recognition that commensal microbes play a key role in resistance to CDI. Current antibiotic treatment of CDI is only partially successful due to a high rate of relapse. As a result, there is interest in understanding the effects of microbes on CDI susceptibility to support treatment of patients with probiotic microbes or entire microbial communities (e.g., fecal microbiota transplantation). The results reported here demonstrate that colonization with the human commensal fungus Candida albicans protects against lethal CDI in a murine model. Colonization with C. albicans did not increase the colonization resistance of the host. Rather, our findings showed that one effect of C. albicans colonization was to enhance a protective immune response. Mice pre-colonized with C. albicans expressed higher levels of IL-17A in infected tissue following C. difficile challenge compared to mice that were not colonized with C. albicans. Administration of cytokine IL-17A was demonstrated to be protective against lethal murine CDI in mice not colonized with C. albicans. C. albicans colonization was associated with changes in the abundance of some bacterial components of the gut microbiota. Therefore, C. albicans colonization altered the gut ecosystem, enhancing survival after C. difficile challenge. These findings demonstrate a new, beneficial role for C. albicans gut colonization.

A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage.

Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O2 conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections.

Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance.

Reactive oxygen species (ROS) generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs), toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs). Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation. Nonetheless, these pathogens often rely on repair and detoxifying proteins in addition to these secreted effectors and toxins in order to resist mammalian sources of ROS. This suggests that pathogens have both intrinsic and extrinsic mechanisms to avoid restriction by PMN-derived ROS. Here, we review mechanisms of oxidative burst in PMNs in response to bacterial infections, as well as the mechanisms by which bacterial pathogens thwart restriction by ROS to survive under conditions of oxidative stress.