Host aging induces a senescent-like phenotype in neutrophils and altered transcriptional responses to .

Aging drives increased susceptibility to respiratory infections by (pneumococci). Polymorphonuclear leukocytes (PMNs) are among the first responders in the lung following pneumococcal infection and are required for bacterial clearance. However, PMN antimicrobial function declines with age.

Large-scale combination screens reveal 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.

Non-canonical autophosphorylation of RIPK1 drives timely pyroptosis to control Yersinia infection.

Caspase-8-dependent pyroptosis has been shown to mediate host protection from Yersinia infection. For this mode of cell death, the kinase activity of receptor-interacting protein kinase 1 (RIPK1) is required, but the autophosphorylation sites required to drive caspase-8 activation have not been determined. Here, we show that non-canonical autophosphorylation of RIPK1 at threonine 169 (T169) is necessary for caspase-8-mediated pyroptosis.

Blocking HXA-mediated neutrophil elastase release during lung infection limits pulmonary epithelial barrier disruption and bacteremia.

(), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread.

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.

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

Unlabelled: 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 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, (Ab), (Kp), and (Pa).

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

Yersinia pseudotuberculosis is an 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).

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, (), and M cells.

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and 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, and . 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, and , and for robust killing of .

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

The 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 gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes.

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.

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

is a respiratory, blood, liver, and bladder pathogen of significant clinical concern. We show that the adaptor protein, SKAP2, is required for protection against (ATCC 43816) pulmonary infections. - mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells.

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

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. has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied.

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.

Unraveling neutrophil- interactions during tissue infection.

The human and animal pathogens , which causes bubonic and pneumonic plague, and and , 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.

Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-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 β-lactamase, are used to infect mice, tissues isolated from mice, or mammalian cells in culture.

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

The emergence of multidrug-resistant has rendered a large array of infections difficult to treat. In a high-throughput genetic screen of factors required for 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 to overcome attenuating mutations in , , , , , , , and in WT and immunosuppressed mice.

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

Over the past few decades, 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 culture have included the development of methods and technology that incorporate tissue structure, cellular composition, and efficient long-term culture.

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.

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.

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.

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.