Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria.

Unlabelled: Pneumococcal pneumonia causes cytotoxicity in the lung parenchyma but the underlying mechanism involves multiple factors contributing to cell death. Here, we discovered that hydrogen peroxide produced by (Spn-H O ) plays a pivotal role by oxidizing hemoglobin, leading to its polymerization and subsequent release of labile heme. At physiologically relevant levels, heme selected a population of encapsulated pneumococci.

Memory Th17 cell-mediated protection against lethal secondary pneumococcal pneumonia following influenza infection.

() frequently causes secondary pneumonia after influenza A virus (IAV) infection, leading to high morbidity and mortality worldwide. Concomitant pneumococcal and influenza vaccination improves protection against coinfection but does not always yield complete protection. Impaired innate and adaptive immune responses have been associated with attenuated bacterial clearance in influenza virus-infected hosts.

Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies.

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase () in survival in a mouse lung infection model. Here, we demonstrate that H.

Antigen-specific memory Th17 cells promote cross-protection against nontypeable Haemophilus influenzae after mild influenza A virus infection.

Secondary bacterial pneumonia after influenza A virus (IAV) infection is the leading cause of hospitalization and death associated with IAV infection worldwide. Nontypeable Haemophilus influenzae (NTHi) is one of the most common causes of secondary bacterial pneumonia. Current efforts to develop vaccines against NTHi infection focus on inducing antibodies but are hindered by antigenic diversity among NTHi strains.

Prophylactic Inhibition of Colonization by Streptococcus pneumoniae with the Secondary Bile Acid Metabolite Deoxycholic Acid.

Streptococcus pneumoniae colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite deoxycholic acid (DoC) affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed the antimicrobial activity of DoC and investigated its potential to eradicate S.

Sialic Acid Protects Nontypeable Haemophilus influenzae from Natural IgM and Promotes Survival in Murine Respiratory Tract.

Nontypeable (NTHi), a common inhabitant of the human nasopharynx and upper airways, causes opportunistic respiratory tract infections that are frequently recurring and chronic. NTHi utilizes sialic acid from the host to evade antibacterial defenses and persist in mucosal tissues; however, the role of sialic acid scavenged by NTHi during infection is not fully understood. We previously showed that sialylation protects specific epitopes on NTHi lipooligosaccharide (LOS) targeted by bactericidal IgM in normal human serum.

A Transcriptional Activator of Ascorbic Acid Transport in Is Required for Optimal Growth in Endophthalmitis in a Strain-Dependent Manner.

is among the top causes of bacterial endophthalmitis, an infectious disease of the intraocular fluids. The mechanisms by which grows and thrives in the intraocular cavity are not well understood. We used a bacterial genome-wide assessment tool (transposon insertion site sequencing) to determine genes essential for growth in vitreous humor.

Underlying Glycans Determine the Ability of Sialylated Lipooligosaccharide To Protect Nontypeable Haemophilus influenzae from Serum IgM and Complement.

Nontypeable (NTHi) efficiently colonizes the human nasopharynx asymptomatically but also causes respiratory mucosal infections, including otitis media, sinusitis, and bronchitis. The lipooligosaccharide (LOS) on the cell surface of NTHi displays complex glycans that mimic host structures, allowing it to evade immune recognition. However, LOS glycans are also targets of host adaptive and innate responses.

Suppression of Alternative Lipooligosaccharide Glycosyltransferase Activity by UDP-Galactose Epimerase Enhances Murine Lung Infection and Evasion of Serum IgM.

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood.

Recognition of conserved antigens by Th17 cells provides broad protection against pulmonary infection.

Nontypeable (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains.

No-Go Zones for Mariner Transposition.

The property of transposons to randomly insert into target DNA has long been exploited for generalized mutagenesis and forward genetic screens. Newer applications that monitor the relative abundance of each transposon insertion in large libraries of mutants can be used to evaluate the roles in cellular fitness of all genes of an organism, provided that transposition is in fact random across all genes. In a recent article, Kimura and colleagues identified an important exception to the latter assumption [S.

Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi.

Outer membrane protein P5 is required for resistance of nontypeable Haemophilus influenzae to both the classical and alternative complement pathways.

The complement system is an important first line of defense against the human pathogen Haemophilus influenzae. To survive and propagate in vivo, H. influenzae has evolved mechanisms for subverting this host defense, most of which have been shown to involve outer surface structures, including lipooligosaccharide glycans and outer surface proteins.

Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung.

Bacterial coinfection represents a major cause of morbidity and mortality in epidemics of influenza A virus (IAV). The bacterium Haemophilus influenzae typically colonizes the human upper respiratory tract without causing disease, and yet in individuals infected with IAV, it can cause debilitating or lethal secondary pneumonia. Studies in murine models have detected immune components involved in susceptibility and pathology, and yet few studies have examined bacterial factors contributing to coinfection.

Genome-scale approaches to identify genes essential for Haemophilus influenzae pathogenesis.

Haemophilus influenzae is a Gram-negative bacterium that has no identified natural niche outside of the human host. It primarily colonizes the nasopharyngeal mucosa in an asymptomatic mode, but has the ability to disseminate to other anatomical sites to cause otitis media, upper, and lower respiratory tract infections, septicemia, and meningitis. To persist in diverse environments the bacterium must exploit and utilize the nutrients and other resources available in these sites for optimal growth/survival.

High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis.

A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.

Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative bacterial pathogen that causes upper and lower respiratory infections. Factors required for pulmonary infection by NTHI are not well understood. Previously, using high-throughput insertion tracking by deep sequencing (HITS), putative lung colonization factors were identified.

High-throughput insertion tracking by deep sequencing for the analysis of bacterial pathogens.

Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis.

ArcA-regulated glycosyltransferase lic2B promotes complement evasion and pathogenesis of nontypeable Haemophilus influenzae.

Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae.

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model.

Resistance of Haemophilus influenzae to reactive nitrogen donors and gamma interferon-stimulated macrophages requires the formate-dependent nitrite reductase regulator-activated ytfE gene.

Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels.

Structure of YraM, a protein essential for growth of Haemophilus influenzae.

Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H.

Identification and analysis of essential genes in Haemophilus influenzae.

The human respiratory pathogen Haemophilus influenzae, a Gram-negative bacterium, is the first free-living organism to have its complete genome sequenced, providing the opportunity to apply genomic-scale approaches to study gene function. This chapter provides an overview of a highly efficient, in vitro mariner transposon-based method that exploits the natural transformation feature of this organism for the identification of essential genes. In addition, we describe strategies for conditional expression systems that would facilitate further analysis of this class of genes.

The periplasmic disulfide oxidoreductase DsbA contributes to Haemophilus influenzae pathogenesis.

Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and causes disease when it invades the bloodstream, lungs, or middle ear. Proteins that mediate critical interactions with the host during invasive disease are likely to be secreted. Many secreted proteins require addition of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability.

The ArcA regulon and oxidative stress resistance in Haemophilus influenzae.

Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H.