REVIEW URRENT C OPINION Haemophilus influenzae: recent advances in the understanding of molecular pathogenesis and polymicrobial infections Farshid Jalalvand and Kristian Riesbeck Purpose of review Non-typeable Haemophilus influenzae (NTHi) is a human-specific mucosal pathogen and one of the most common causes of bacterial infections in children and patients with chronic obstructive pulmonary disease. It is also frequently found in polymicrobial superinfections. Great strides have recently been made in the understanding of the molecular mechanisms underlying NTHi pathogenesis. Recent findings By using new methodology, such as experimental human colonization models and whole-genome approaches, investigators have shed light upon the various strategies of NTHi that are involved in pathogenesis. These include the escape of the mucociliary elevator, evasion of host immunity, survival in environments with scarce nutrients, and finally participation in polymicrobial infections. Lipooligosaccharide branching, proteinous adhesins, metabolic adaption to nutrient availability and many scavenging systems are implicated in these processes. Interestingly, genome-based studies comparing virulent and commensal strains have identified many hypothetical proteins as virulence determinants, suggesting that much regarding the molecular pathogenesis of NTHi remains to be solved. Summary NTHi is an opportunistic pathogen and highly specialized colonizer of the human respiratory tract that has developed intricate mechanisms to establish growth and survival in the human host. Continued research is needed to further elucidate NTHi host–pathogen and pathogen–pathogen interactions. Keywords Haemophilus influenzae, immune evasion, molecular pathogenesis, niche adaptation, polymicrobial infections INTRODUCTION Haemophilus influenzae is a commensalistic Gramnegative species of the human upper respiratory tract microbiota and a frequent cause of mucosal infections . As a model organism, H. influenzae has been involved in several major scientific achievements throughout history; it was the source of the isolation of the first restriction enzyme , it was used to develop the first animal model for bacterial meningitis  and was also the first free-living organism to have its genome fully sequenced . Furthermore, the first licensed glycoconjugate vaccine for use in humans was developed against encapsulated H. influenzae type b (Hib) , and, more recently, H. influenzaederived protein D has successfully been employed as the carrier protein for a licensed pneumococcal conjugate vaccine . www.co-infectiousdiseases.com Non-typeable, that is, uncapsulated H. influenzae (NTHi) is a prevalent cause of acute and recurrent otitis media in children and exacerbations in chronic obstructive pulmonary disease (COPD) patients [7–11]. It is also the etiological agent of bacterial pulmonary infections in immunocompromised hosts such as lung cancer patients, and occasionally disseminates to cause invasive disease [11–14]. To establish a successful colonization, Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden Correspondence to Dr Kristian Riesbeck, Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Jan Waldenströms gata 59, SE-205 02 Malmö, Sweden. Tel: +46 40 338494; e-mail: firstname.lastname@example.org Curr Opin Infect Dis 2014, 27:268–274 DOI:10.1097/QCO.0000000000000056 Volume 27 Number 3 June 2014 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Haemophilus influenzae pathogenesis Jalalvand and Riesbeck KEY POINTS NTHi causes mucosal and invasive opportunistic infections in humans to a high socioeconomic cost. NTHi is associated with a large degree of genetic heterogeneity and interstrain DNA exchange. NTHi is highly adapted to colonize the human respiratory tract and evades swift clearance by the host immune system via virulence factors including lipo-oligosaccharide, proteinous adhesins and binding of complement inhibitors. NTHi has developed intricate systems to metabolically respond to changes in the microenvironment. Recent studies have elucidated some of the molecular mechanisms underlying polymicrobial infections involving NTHi and co-pathogens, and these include viral attenuation of the host immune response. NTHi has to cope with the structural barrier of the mucociliary epithelium, the innate and acquired immunity, intermicrobial competition, and nutrient poor environments. NTHi is highly adapted to these conditions and usually establishes a transient colonization with a turnover rate of 3 months; the host is overtaken by a new strain as the immune system clears the preceding one . As NTHi currently causes the vast majority of H. influenzae infections, this review is primarily focused on recent advances related to the molecular pathogenesis of NTHi. GENETIC INTRASPECIES HETEROGENEITY H. influenzae is a genetically diverse species with each strain containing between 1765 and 2355 genes (http://www.ncbi.nlm.nih.gov/genome/genomes/ 165). The core-genome present in all strains consists of 1485 genes, or about 75% of the genomic content of any given isolate [16 ]. The supragenome of H. influenzae, however, has been predicted to contain approximately 4500 unique genes. As many strains are naturally competent, it is plausible that a constant and dynamic genetic exchange occurs via uptake and recombination of intraspecies DNA containing unique H. influenzae uptake sequences , with individual strains having access to various parts of the supragenome during concomitant colonization of the same host [18 ,19]. This mechanism would allow the bacteria to access a wide variety of genes while still keeping their individual genomes small, thus giving them a fitness advantage. Specific disease-associated genetic elements, as opposed to asymptomatic colonization factors, have && & long remained elusive, although some genes have been found to be more prevalent among virulent strains, as has been reviewed . However, when the complete gene content of 210 geographically and clinically diverse NTHi strains was recently compared, 149 genes were identified to be significantly associated with either virulence or commensalism [16 ]. Interestingly, the 28 genes that were more likely to be found in virulent strains were not any of the well characterized virulence factors involved in adherence, lipo oligosaccharide (LOS) biosynthesis, or immune evasion. Indeed, most were hypothetical proteins indicating that the principal virulence determinants may have eluded researchers thus far. && HOST COLONIZATION AND PERSISTENCE The initial step of successful colonization and subsequent infection is adherence to the host tissue. To circumvent mechanical clearance by the mucosal epithelium, NTHi uses four main strategies: perturbance of the mucociliary elevator, adherence to the epithelium, formation of microcolonies and biofilm, and/or invasion of epithelial cells. One mechanism underlying NTHi-mediated decreased ciliary beating and detachment of ciliated cells was recently shown to involve the activation of host protein kinase C epsilon . However, disintegrity of ciliated cells is not always observed, as reported by other investigators using extended time co-culture (1–10 days) with NTHi and primary human respiratory epithelial cells . Instead, bacteria appeared to seek refuge in paracellular foci to evade the mucociliary elevator. Intercellular bacteria were seemingly associated with the basal cell layers, where junctional disorganization was observed. Both these strategies can plausibly be implemented during human colonization. Epithelial cells are connected to the structural scaffold of the underlying extracellular matrix (ECM) via a range of basal surface structures including integrins . Should these interactions be disrupted (as observed in the studies above), or the epithelium be damaged by viral infections or chronic inflammation, ECM proteins become a viable target for adherence by pathogens . Like other respiratory tract pathogens, NTHi has evolved strategies to bind to ECM proteins. We recently reported that NTHi protein F, a homolog of streptococcal laminin-binding proteins Lbp/Lmb, is a novel adhesin that directly mediates bacterial binding to laminin and primary human bronchial epithelial cells [25 ]. These results are in analogy with NTHi adhesins protein E and Hap that have previously been reported to interact with both ECM proteins as 0951-7375 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins & www.co-infectiousdiseases.com 269 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Pathogenesis and immune response well as host cells [26–28]. Interestingly, protein F (gene also annotated as hfeA) has been found to be crucial for successful NTHi infection in vivo [29 ]. Animal models with mice, rats, and chinchillas have traditionally been used to mimic human NTHi infections, but the relevance of the data obtained in these studies will always remain an issue because of the human-specific nature of the pathogen. In a new and exciting study, Winokur et al. [30 ] developed a human nasopharyngeal colonization model to study the virulence of NTHi in its natural host. Thirteen selected phase-variable genes were monitored during a 6-day period of human colonization and significant changes were observed in two genes, phosphorylcholine kinase licA and IgA1-protease igaB [31 ]. LicA incorporates exogenously acquired phosphorylcholine into LOS, an alteration that mediates increased adherence and entry into bronchial epithelial cells. The in-vivo selection of subpopulations shifting to phase-on expression of licA suggests that this mechanism is of importance during early colonization. Invasion and persistence of NTHi in epithelial cells have been extensively reviewed . There is also increasing evidence showing that NTHi may reside in lymphoid tissues and act as a reservoir for recurrent infections [33,34]. Additional mechanisms of H. influenzae persistence are reported to involve classic toxin–antitoxin systems that may arrest cell proliferation during stressful conditions via mRNA targeting and result in dormant persister cells . In a recent report, NTHi IgA1 proteases were implicated in internalization and intracellular persistence in bronchial epithelial cells . IgA1 protease is a secreted virulence factor with high specificity for the predominant mucosal immunoglobulin, secretory IgA1, which it cleaves in the hinge region to circumvent the host humoral response. As a testament to its importance for pathogenesis, the protease was one of two phase-variable genes shown to be significantly upregulated during experimental human colonization [31 ]. Two variants of IgA1 protease have been identified in NTHi, IgaA and, more recently, IgaB . Authors found that IgaA was required for optimal invasion of epithelial cells, whereas IgaB was needed for optimal intracellular persistence. Intriguingly, it is unclear how IgaA enhances NTHi internalization. Possible mechanisms could be a yet unidentified enzymatic function that the secreted protease exerts on bacterial and/or host cells, or interaction between the outer membrane-integrated residual IgaA translocator domain and epithelial cells. Albeit previously controversial, the formation of NTHi biofilm in vivo is nowadays generally acknowledged by most researchers. NTHi residing in && && && && 270 www.co-infectiousdiseases.com biofilms, as compared with planktonic subsistence, acquire a series of advantages including enhanced resistance to antimicrobial compounds and host immune effectors [37,38 ]. The current understanding of NTHi biofilms has recently been excellently reviewed in several publications [37,38 ,39,40]. && && NICHE ADAPTABILITY AND NUTRIENT ACQUISITION The human nasopharynx is a nutrient-poor environment for bacteria, especially during inflammation when the host sequesters essential nutrients [41 ]. To establish a successful colonization of the respiratory tract, otopathogens rely on their ability to adapt to this niche, scavenge scarce nutrients, and respond metabolically to changes in the environment. A recent study found that H. influenzae carbonic anhydrase is essential for growth in low CO2 concentrations and important for prolonged intracellular survival in phagosomes, suggesting a role for this enzyme in bacterial adaption to different CO2 conditions and acidic pH . The pathogen also utilizes urease, found to be more prevalent in disease-causing isolates than commensals, to survive in acid environments [43,44]. Other systems involved in sensing the microenvironment within the host include the multifunctional Sap-transporter, possibly via detection of host molecules as well as heme accessibility [45,46]. As alluded to by its name, Haemophilus lacks the ability to synthesize heme that is essential for its survival. Heme is thus acquired exogenously and the pathogen has developed many ways for sensing extracellular heme (and iron) and altering its gene expression to correctly respond to the availability of the metabolite [47,48]. Expectedly, disruption of these systems results in decreased virulence [46–48]. One of the central transcriptional regulators involved in iron-acquisition is the ferric uptake regulator (Fur) protein. The Fur regulon of NTHi was recently defined and shown to contain 73 genes . Interestingly, authors found that in addition to iron uptake and utilization genes, IgA1 protease was also regulated by Fur, suggesting that the bacterium has coupled low iron concentrations to the mucosal milieu. An isogenic Dfur mutant was found to be attenuated in vivo. Fur is not the only regulatory responder to low iron concentrations. Recently, the core modulon (identical in five different isolates) of H. influenzae adaptation to low levels of iron/heme was defined in vitro and verified in vivo [50 ]. In addition to the 55 genes that were similarly regulated in all strains, authors identified approximately 200 non-core & & Volume 27 Number 3 June 2014 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Haemophilus influenzae pathogenesis Jalalvand and Riesbeck genes that they hypothesize are important for environmental adaptation. The conditions in the lower respiratory tract of COPD patients differ significantly from those in the nasopharynx, with mucus hypersecretion (that probably causes hypoxic microenvironments), failure of cilia-mediated clearance, high cytokine levels, excessive phagocyte presence, epithelial disintegrity, and oxidant/antioxidant imbalances . The genetic adaptation of NTHi populations to these changes have been examined and 15 genetic islands have been identified to be more prevalent in COPD isolates as compared with commensal nasopharyngeal strains, including genes encoding the IgA1 protease, high-molecular-weight adhesins, and urease . Interestingly, some of these genes had previously been observed to be transcriptionally upregulated during bacterial growth in sputum obtained from COPD patients, suggesting in-situ selection for both genetically and transcriptionally adapted strains . Other factors that potentially contribute to the etiology of exacerbations in COPD patients include bacterial oxidative stress responses and impaired host defenses targeting NTHi [53,54]. The topic of reactive oxygen species-resisting NTHi mechanisms has been thoroughly reviewed recently [55 ]. & INFLAMMATION AND IMMUNE EVASION In recent years, great achievements have been made in the molecular understanding of NTHi-mediated inflammation processes during otitis media and COPD exacerbations [56,57]. The pathogenesis differs in these conditions, with the balance being shifted toward immune suppression and immune activation in otitis media and COPD, respectively. Nevertheless, evading the host innate immunity, particularly the complement system, is essential for all human bacterial pathogens . NTHi relies on two main strategies for complement evasion: camouflage using branched LOS that covers the bacterial surface or binding of host complement regulator proteins. By attaching a variety of compounds to the LOS core, including scavenged host-specific sialic acid and phosphorylcholine, NTHi blocks the accessibility of bactericidal antibodies to its other surface structures, thus inhibiting complement activation via the classical pathway [59,60 ,61 ,62,63]. Interestingly, this mechanism has shown to be induced in vivo and during exposure to human serum [31 ,60 ,61 ,62]. Other host– pathogen interactions that LOS has been implicated in include biofilm formation and resistance to antimicrobial peptides [64 ]. We have previously shown that acquisition of the host complement inhibitor vitronectin is & & && && & & important for NTHi serum resistance. By using a proteomic approach to identify vitronectin-binding NTHi proteins, we found that Haemophilus protein F mediates complement evasion via sequestration of vitronectin at the bacterial surface [65 ]. & POLYMICROBIAL INFECTIONS Recent insight into bacterial cell–cell interactions has shown stunning displays of specific and speciesunique secretory responses to direct stimuli [66 ]. It is therefore not surprising that mixed pneumococcal/NTHi otitis media are suggested to be clinically distinct infections compared with single organism otitis media . Co-colonization/infections have long been observed with the otopathogens NTHi, pneumococci, and Moraxella catarrhalis [68–74], but some studies have also reported antagonistic relationships between them [75,76]. Moreover, secondary NTHi infections are commonly detected in patients infected by viral pathogens such as influenza A virus (IAV), rhinovirus, and human respiratory syncytial virus [71,77–80]. It could be that NTHi is particularly well adapted to interplay with other bacteria as well as to host microenvironments affected by primary viral infections. The understanding of the molecular mechanisms underlying polymicrobial superinfections has improved during the last years. One such mechanism was recently unfolded as investigators showed that rhinovirus-induced degradation of the host signaling pathway adaptor protein IRAK-1 attenuates the TLR2-mediated response to NTHi [81 ]. This results in delayed neutrophil recruitment that hinders swift bacterial clearance, providing one explanation as to why rhinoviral/NTHi superinfections are frequently seen clinically. We recently shed light on the physiological interplay between NTHi and group A streptococci (GAS), two pathogens whose presence has been shown to correlate in children suffering from acute pharyngotonsillitis [82,83 ]. We showed that NTHiderived outer membrane vesicles (OMVs) carried functional b-lactamase that protected GAS from amoxicillin-mediated killing. Interestingly, NTHi OMV shedding is seen in several infection models, suggesting that they may play important roles during pathogenesis [22,45]. In a seminal article published by Wong et al. [29 ], investigators employed transposon insertion sequencing to define the NTHi genes needed during single organism infection, co-infection with IAV, and the core set of genes required during both conditions. In addition to observing that IAV made mice highly susceptible to NTHi infections, authors noted that several (but not all) bacterial genes 0951-7375 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins && && & && www.co-infectiousdiseases.com 271 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Pathogenesis and immune response involved in defense against various stress conditions were dispensable in co-infections, suggesting that IAV (like rhinovirus) attenuates the host immune response to NTHi. Other interesting findings included the bacterial adaptation to the changes in nutrient access brought on by the viral infection, including alterations in stress responses. These data provide valuable insight in to the idiosyncrasies of polymicrobial infections, and the continued elucidation of the dynamic molecular interactions augmenting NTHi disease in polymicrobial settings will be highly interesting to follow. Conflicts of interest There are no conflicts of interest. REFERENCES AND RECOMMENDED READING CONCLUSION NTHi is a remarkable pathogen in many aspects. In contrast to many of its co-pathogens, NTHi does not have any known cytotoxic effectors or exotoxins, secreted proteolytic enzymes (aside from the IgA1specific endopeptidases), lipases, super antigens, injectisome secretion systems, or a polysaccharide capsule. Still it manages to efficiently outcompete other microbes and colonize the upper respiratory tract of children and the lower respiratory tract of immunocompromised adults. How does it do it? NTHi has developed advanced and efficient scavenging systems for obtaining nutrients in the human airways. Indeed, it has been reported that the pathogen produces siderophore-utilization proteins without producing siderophores , meaning it steals iron from other bacterial species that in turn steal it from the host! Furthermore, NTHi scavenges essential and host-specific nutrients, effectively saving the cost of synthesizing these products. The price of the loss of biosynthesis enzymes is niche dependency, as NTHi can only sustain prolonged growth in the human respiratory tract. We have gained increasing insight into other virulence mechanisms of NTHi. Investigators have shown that NTHi employs an array of multifunctional proteins, including protein D, protein E, protein F, the Sap-transporter and the heme-binding proteins HbpA and TehB, a display of highly economic use of proteins [25 ,26,27,45,46,65 ,85– 89]. Moreover, genetic heterogeneity and accessibility to a diverse supragenome has provided NTHi with additional fitness benefits as each isolate can maintain a relatively small genome and still be adaptable to environmental selection [16 ]. To summarize, NTHi is a highly adapted human-specific commensal whose molecular pathogenesis is not yet fully understood. The clinical burden of this opportunistic pathogen encourages more research to help combat infections caused by it. & & && 272 Acknowledgements This work was supported by grants from the Alfred Österlund, the Anna and Edwin Berger, Greta and Johan Kock foundations, the Swedish Medical Research Council (grant number 521-2010-4221, www.vr.se), the Physiographical Society (Forssman’s Foundation), and Skåne County Council’s research and development foundation. www.co-infectiousdiseases.com Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. King P. Haemophilus influenzae and the lung (Haemophilus and the lung). Clin Transl Med 2012; 1:10. 2. Smith HO, Wilcox KW. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 1970; 51:379–391. 3. Smith AL, Smith DH, Averill DR Jr, et al. 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