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

Streptococcus pneumoniae (Sp) 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. In this study, we showed that preceding low-dose IAV infection caused persistent Sp infection and suppression of bacteria-specific T-helper type 17 (Th17) responses in mice. Prior Sp infection protected against subsequent IAV/Sp coinfection by improving bacterial clearance and rescuing bacteria-specific Th17 responses in the lungs. Furthermore, blockade of IL-17A by anti-IL-17A antibodies abrogated the protective effect of Sp preinfection. Importantly, memory Th17 responses induced by Sp preinfection overcame viral-driven Th17 inhibition and provided cross-protection against different Sp serotypes following coinfection with IAV. These results indicate that bacteria-specific Th17 memory cells play a key role in providing protection against IAV/Sp coinfection in a serotype-independent manner and suggest that a Th17-based vaccine would have excellent potential to mitigate disease caused by coinfection. IMPORTANCE Streptococcus pneumoniae (Sp) frequently causes secondary bacterial pneumonia after influenza A virus (IAV) infection, leading to increased morbidity and mortality worldwide. Current pneumococcal vaccines induce highly strain-specific antibody responses and provide limited protection against IAV/Sp coinfection. Th17 responses are broadly protective against Sp single infection, but whether the Th17 response, which is dramatically impaired by IAV infection in naïve mice, might be effective in immunization-induced protection against pneumonia caused by coinfection is not known. In this study, we have revealed that Sp-specific memory Th17 cells rescue IAV-driven inhibition and provide cross-protection against subsequent lethal coinfection with IAV and different Sp serotypes. These results indicate that a Th17-based vaccine would have excellent potential to mitigate disease caused by IAV/Sp coinfection.

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 (copA) in survival in a mouse lung infection model. Here, we demonstrate that H. influenzae copA (HI0290) is responsible for copper homeostasis involving the merR-type regulator, cueR, as well as six tandem copies of the metallochaperone gene, copZ. Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. Nontypeable H. influenzae (NTHi) clinical isolate NT127 has the same locus organization but with three copies of copZ. We showed that expression of the NTHi copZA operon is activated by copper under the regulatory control of CueR. NTHi single copA and copZ mutants and, especially, the double deletion copZA mutant exhibited decreased copper tolerance, and the ΔcopZA mutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase (copA) alone and deleted of both the ATPase and chaperones (copZ1-3) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation of cop locus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that the cop system encodes an important countermeasure to alleviate copper toxicity.

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 in vitro the antimicrobial activity of DoC and investigated its potential to eradicate S. pneumoniae colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration, DoC (0.5 mg/ml; 1.27 mM) killed all tested S. pneumoniae strains (n = 48) 2 h postinoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by S. pneumoniae strains as soon as 10 min postexposure. The mechanism of action did not involve activation of autolysis, since the autolysis-defective double mutants ΔlytAΔlytC and ΔspxBΔlctO were as susceptible to DoC as was the wild type (WT). Oral streptococcal species (n = 20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1 × 10-9, no spontaneous resistance was observed with DoC (srF, ≥1 × 10-12). Finally, the efficacy of DoC to eradicate S. pneumoniae colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with S. pneumoniae EF3030 carried a median of 4.05 × 105 CFU/ml 4 days postinoculation compared to 6.67 × 104 CFU/ml for mice treated with DoC. Mice in the prophylactic group had an ∼99% reduction of the pneumococcal density (median, 2.61 × 103 CFU/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against S. pneumoniae.

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

Nontypeable Haemophilus influenzae (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. Here, we evaluated the importance of immune evasion mediated by LOS sialylation in the mouse respiratory tract using wild-type H. influenzae and an isogenic siaB mutant incapable of sialylating the LOS. Sialylation protected common NTHi glycan structures recognized by human and murine IgM and protected NTHi from complement-mediated killing directed by IgM against these structures. Protection from IgM binding by sialylated LOS correlated with decreased survival of the siaB mutant versus the wild type in the murine lung. Complement depletion with cobra venom factor increased survival of the siaB mutant in the nasopharynx but not in the lungs, suggesting differing roles of sialylation at these sites. Prior infection increased IgM against H. influenzae but not against sialic acid-protected epitopes, consistent with sialic acid-mediated immune evasion during infection. These results provide mechanistic insight into an NTHi evasive strategy against an immune defense conserved across host species, highlighting the potential of the mouse model for development of anti-infective strategies targeting LOS antigens of NTHi.

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

Streptococcus pneumoniae is among the top causes of bacterial endophthalmitis, an infectious disease of the intraocular fluids. The mechanisms by which S. pneumoniae 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 S. pneumoniae growth in vitreous humor. The results indicated that an ascorbic acid (AA) transport system subunit was important for growth. We created an isogenic gene deletion mutant of the AA transcriptional activator, ulaR2, in 2 strains of S. pneumoniae. Growth curve analysis indicated that ulaR2 deletion caused attenuated growth in vitro for both strains. However, in vivo vitreous humor infection in rabbits with either strain determined that ulaR2 was necessary for growth in one strain but not the other. These results demonstrate that ulaR2 may be important for fitness during S. pneumoniae endophthalmitis depending on the background of the strain.

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

Nontypeable Haemophilus influenzae (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. To aid in evasion of these responses, LOS structures exhibit interstrain heterogeneity and are also subject to phase variation, the random on/off switching of gene expression, generating intrastrain population diversity. Specific LOS modifications, including terminal sialylation of the LOS, which exploits host-derived sialic acid (Neu5Ac), can also block recognition of NTHi by bactericidal IgM and complement by mechanisms that are not fully understood. We investigated the LOS sialic acid-mediated resistance of NTHi to antibody-directed killing by serum complement. We identified specific LOS structures extending from heptose III that are targets for binding by naturally occurring bactericidal IgM in serum and are protected by sialylation of the LOS. Phase-variable galactosyltransferases encoded by lic2A and lgtC each add a galactose epitope bound by IgM that results in antibody-dependent killing via the classical pathway of complement. NTHi's survival can be influenced by the expression of phase-variable structures on the LOS that may also depend on environmental conditions, such as the availability of free sialic acid. Identification of surface structures on NTHi representing potential targets for antibody-based therapies as alternatives to antibiotic treatment would thus be valuable for this medically important pathogen.

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. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE. Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

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

Nontypeable Haemophilus influenzae (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. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches.

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. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial.

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. We conducted genome-wide profiling of the H. influenzae genes that promote its fitness in a murine model of coinfection with IAV. Application of direct, high-throughput sequencing of transposon insertion sites revealed fitness phenotypes of a bank of H. influenzae mutants in viral coinfection in comparison with bacterial infection alone. One set of virulence genes was required in nonvirally infected mice but not in coinfection, consistent with a defect in anti-bacterial defenses during coinfection. Nevertheless, a core set of genes required in both in vivo conditions indicated that many bacterial countermeasures against host defenses remain critical for coinfection. The results also revealed a subset of genes required in coinfection but not in bacterial infection alone, including the iron-sulfur cluster regulator gene, iscR, which was required for oxidative stress resistance. Overexpression of the antioxidant protein Dps in the iscR mutant restored oxidative stress resistance and ability to colonize in coinfection. The results identify bacterial stress and metabolic adaptations required in an IAV coinfection model, revealing potential targets for treatment or prevention of secondary bacterial pneumonia after viral infection.

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. Recent evidence suggests that regulatory factors that direct such adaptations also control virulence determinants required to resist and evade immune clearance mechanisms. In this review, we describe the recent application of whole-genome approaches that together provide insight into distinct survival mechanisms of H. influenzae in the context of different sites 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. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.

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. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.

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. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

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. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-gamma)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-gamma or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

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. Finally, we outline a method based on the approach used in H. influenzae for identifying essential genes that can be applied to other bacteria that are not naturally transformable.

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. In this study, we evaluated the role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models, and complemented strains exhibited virulence equivalent to that of the parental strains. Comparison of predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was examined because of the importance of exogenous heme for aerobic growth of H. influenzae. The presence of a dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and complementation restored its in vivo phenotype to that of the parental strain. These results indicate that dsbA is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H. influenzae pathogenesis.

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. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H(2)O(2) resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection.

Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide.

In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence-associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS-PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.

SCE jumping: genetic tool for allelic exchange in bacteria.

As more microbial genome sequence information becomes available, the field of bacterial pathogenesis would benefit from the development of new genetic tools designed to facilitate gene function studies on a genomic scale. The complete DNA sequence of the bacterium Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major opportunistic human pathogen. Here, I describe the development of a new gene replacement scheme termed "SCE jumping" in P. aeruginosa. The system uses the yeast I-SceI homing endonuclease in conjunction with in vitro mariner-transposon mutagenesis to generate mutations within targeted regions of the chromosome for genetic footprinting. Use of SCE jumping for generating transposon insertion mutants is anticipated to be widely applicable to other bacterial organisms. This allelic exchange strategy is discussed in context with other methods of gene replacement strategies available in P. aeruginosa. Development of SCE jumping provides an excellent example of the power of importing systems from unrelated organisms to circumvent practical challenges in molecular genetics.