Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.

A Humanized Monoclonal Antibody Potentiates Killing of Diverse Biofilm-Forming Respiratory Tract Pathogens by Antibiotics.

New strategies to treat diseases in which biofilms contribute significantly to pathogenesis are needed, as biofilm-resident bacteria are highly recalcitrant to antibiotics due to physical biofilm architecture and a canonically quiescent metabolism, among many additional attributes. We, and others, have shown that when biofilms are dispersed or disrupted, bacteria released from biofilm residence are in a distinct physiologic state that, in part, renders these bacteria highly sensitive to killing by specific antibiotics. We sought to demonstrate the breadth of the ability of a recently humanized monoclonal antibody against an essential biofilm structural element (DNABII protein) to disrupt biofilms formed by respiratory tract pathogens and potentiate antibiotic-mediated killing of bacteria released from biofilm residence. Biofilms formed by six respiratory tract pathogens were significantly disrupted by the humanized monoclonal antibody in a dose- and time-dependent manner, as corroborated by confocal laser scanning microscopy (CLSM) imaging. Bacteria newly released from the biofilms of 3 of 6 species were significantly more sensitive than their planktonic counterparts to killing by 2 of 3 antibiotics currently used clinically and were now also equally as sensitive to killing by the 3rd antibiotic. The remaining 3 pathogens were significantly more susceptible to killing by all 3 antibiotics. A humanized monoclonal antibody directed against protective epitopes of a DNABII protein effectively released six diverse respiratory tract pathogens from biofilm residence in a phenotypic state that was now as, or significantly more, sensitive to killing by three antibiotics currently indicated for use clinically. These data support this targeted, combinatorial, species-agnostic therapy to mitigate chronic bacterial diseases.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery.

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

A Protein E-PilA Fusion Protein Shows Vaccine Potential against Nontypeable Haemophilus influenzae in Mice and Chinchillas.

PE-PilA is a fusion protein composed of immunologically relevant parts of protein E (PE) and the majority subunit of the type IV pilus (PilA), two major antigens of nontypeable Haemophilus influenzae (NTHi). Here we report on the preclinical evaluation of PE-PilA as a vaccine antigen. The immunogenic potential of the PE and PilA within the fusion was compared with that of isolated PE and PilA antigens. When injected intramuscularly into mice, the immunogenicity of PE within the fusion was equivalent to that of isolated PE, except when it was formulated with alum. In contrast, in our murine models PilA was consistently found to be more immunogenic as a subentity of the PE-PilA fusion protein than when it was injected as an isolated antigen. Following immunization with PE-PilA, anti-PE antibodies demonstrated the same capacity to inhibit the binding of PE to vitronectin as those induced after PE immunization. Likewise, PE-PilA-induced anti-PilA antibodies inhibited the formation of NTHi biofilms and disrupted established biofilms in vitro These experiments support the immunogenic equivalence between fused PE-PilA and isolated PE and PilA. Further, the potential of PE-PilA immunization against NTHi-induced disease was evaluated. After intranasal NTHi challenge, colonization of the murine nasopharynx significantly dropped in animals formerly immunized with PE-PilA, and in chinchillas, signs of otitis media were significantly reduced in animals that had received anti-PE-PilA antibodies. Taken together, our data support the use of PE-PilA as an NTHi vaccine antigen.

The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media.

BACKGROUND: Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1-6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. RESULTS: Phase-variable expression of modM3, mediated by variation in length of a 5'-(CAAC)n-3' tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5'-ACm6ATC-3'. Methylation was detected at all 4446 5'-ACATC-3' sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. CONCLUSIONS: Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

Targeting the HUß Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms.

The oral cavity is home to a wide variety of bacterial species, both commensal, such as various streptococcal species, and pathogenic, such as Porphyromonas gingivalis, one of the main etiological agents of periodontal disease. Our understanding of how these bacteria ultimately cause disease is highly dependent upon understanding how they coexist and interact with one another in biofilm communities and the mechanisms by which biofilms are formed. Our research has demonstrated that the DNABII family of DNA-binding proteins are important components of the extracellular DNA (eDNA)-dependent matrix of bacterial biofilms and that sequestering these proteins via protein-specific antibodies results in the collapse of the biofilm structure and release of the resident bacteria. While the high degree of similarity among the DNABII family of proteins has allowed antibodies derived against specific DNABII proteins to disrupt biofilms formed by a wide range of bacterial pathogens, the DNABII proteins of P. gingivalis have proven to be antigenically distinct, allowing us to determine if we can use anti-P. gingivalis HUß antibodies to specifically target this species for removal from a mixed-species biofilm. Importantly, despite forming homotypic biofilms in vitro, P. gingivalis must enter preexisting biofilms in vivo in order to persist within the oral cavity. The data presented here indicate that antibodies derived against the P. gingivalis DNABII protein, HUß, reduce by half the amount of P. gingivalis organisms entering into preexisting biofilm formed by four oral streptococcal species. These results support our efforts to develop methods for preventing and treating periodontal disease.IMPORTANCE Periodontitis is one of the most prevalent chronic infections, affecting 40 to 50% of the population of the United States. The root cause of periodontitis is the presence of bacterial biofilms within the gingival space, with Porphyromonas gingivalis being strongly associated with the development of the disease. Periodontitis also increases the risk of secondary conditions and infections such as atherosclerosis and infective endocarditis caused by oral streptococci. To induce periodontitis, P. gingivalis needs to incorporate into preformed biofilms, with oral streptococci being important binding partners. Our research demonstrates that targeting DNABII proteins with an antibody disperses oral streptococcus biofilm and prevents P. gingivalis entry into oral streptococcus biofilm. These results suggest potential therapeutic treatments for endocarditis caused by streptococci as well as periodontitis.

The HMW2 adhesin of non-typeable Haemophilus influenzae is a human-adapted lectin that mediates high-affinity binding to 2-6 linked N-acetylneuraminic acid glycans.

Non-typeable Haemophilus influenzae (NTHi) is a human-adapted bacterial pathogen, responsible for infections of the human respiratory tract. This pathogen expresses a range of adhesins that mediate binding to host cells. Most NTHi strains can express the related adhesins HMW1 and HMW2. Expression of HMW proteins is phase-variable: changes in the length of simple-sequence repeats located in the encoding genes promoter regions results in changes in expression levels of these adhesins. HMW expression is also controlled by epigenetic regulation. HMW1 has been previously demonstrated to bind α 2-3 sialyl-lactosamine, but affinity of this interaction has not been investigated. The host receptor(s) for HMW2 is currently unknown. We hypothesized that host glycans may act as receptors for HMW2-mediated adherence. We examined the glycan-binding activity of HMW2 using glycan arrays and Surface Plasmon Resonance (SPR). These studies demonstrate that HMW2 binds 2-6 linked N-acetylneuraminic acid with high affinity. HMW2 did not bind glycan structures containing the non-human form of sialic acid, N-glycolylneuraminic acid. Thus, the specificity of HMW1 and HMW2 have complementary lectin activities that may allow NTHi distinct niches in the human host.

What's on the Outside Matters: The Role of the Extracellular Polymeric Substance of Gram-negative Biofilms in Evading Host Immunity and as a Target for Therapeutic Intervention.

Biofilms are organized multicellular communities encased in an extracellular polymeric substance (EPS). Biofilm-resident bacteria resist immunity and antimicrobials. The EPS provides structural stability and presents a barrier; however, a complete understanding of how EPS structure relates to biological function is lacking. This review focuses on the EPS of three Gram-negative pathogens: Pseudomonas aeruginosa, nontypeable Haemophilus influenzae, and Salmonella enterica serovar Typhi/Typhimurium. Although EPS proteins and polysaccharides are diverse, common constituents include extracellular DNA, DNABII (DNA binding and bending) proteins, pili, flagella, and outer membrane vesicles. The EPS biochemistry promotes recalcitrance and informs the design of therapies to reduce or eliminate biofilm burden.

Intercellular adhesion molecule 1 serves as a primary cognate receptor for the Type IV pilus of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) utilizes the Type IV pilus (Tfp) to adhere to respiratory tract epithelial cells thus colonizing its human host; however, the host cell receptor to which this adhesive protein binds is unknown. From a panel of receptors engaged by Tfp expressed by other bacterial species, we showed that the majority subunit of NTHI Tfp, PilA, bound to intercellular adhesion molecule 1 (ICAM1) and that this interaction was both specific and of high affinity. Further, Tfp-expressing NTHI inoculated on to polarized respiratory tract epithelial cells that expressed ICAM1 were significantly more adherent compared to Tfp-deficient NTHI or NTHI inoculated on to epithelial cells to which ICAM1 gene expression was silenced. Moreover, pre-incubation of epithelial cells with recombinant soluble PilA (rsPilA) blocked adherence of NTHI, an outcome that was abrogated by admixing rsPilA with ICAM1 prior to application on to the target cells. Epithelial cells infected with adenovirus or respiratory syncytial virus showed increased expression of ICAM1; this outcome supported augmented adherence of Tfp-expressing NTHI. Collectively, these data revealed the cognate receptor for NTHI Tfp as ICAM1 and promote continued development of a Tfp-targeted vaccine for NTHI-induced diseases of the airway wherein upper respiratory tract viruses play a key predisposing role.

ModA2 Phasevarion Switching in Nontypeable Haemophilus influenzae Increases the Severity of Experimental Otitis Media.

Several human-adapted bacterial pathogens use a phasevarion (ie, a phase-variable regulon) to rapidly and reversibly regulate the expression of many genes, which include known virulence factors, yet the influence of phasevarion-mediated regulation in pathogenesis remains poorly understood. Here we examine the impact of the nontypeable Haemophilus influenzae (NTHI) ModA2 phasevarion on pathogenesis and disease severity in a chinchilla model of experimental otitis media. Chinchillas were challenged with NTHI variant populations that were either inoculated ON and remained ON, inoculated OFF and shifted ON, or inoculated OFF and remained OFF, within the middle ear. We show that populations that shift from OFF to ON within the middle ear induce significantly greater disease severity than populations that are unable to shift. These observations support the importance of phasevarion switching in NTHI pathogenesis and the necessity to considered phasevarion regulation when developing methods to treat and prevent infection.

Type IV Pilus Expression Is Upregulated in Nontypeable Haemophilus influenzae Biofilms Formed at the Temperature of the Human Nasopharynx.

UNLABELLED: Nontypeable Haemophilus influenzae (NTHI), a commensal of the human nasopharynx (hNP), is a common cause of biofilm-associated diseases of the respiratory tract. However, NTHI biofilm biology at the average hNP temperature, i.e., 34°C, has not been well studied. Here we grew NTHI biofilms at 34°C and 37°C, to evaluate relative biofilm growth, expression, and function of the type IV pilus (Tfp), a critical adhesin important for NTHI biofilm formation. The kinetics and regulation of Tfp expression in NTHI biofilms are unclear, especially at 34°C. Tfp expression, as estimated by pilA promoter activity, was distributed throughout the biofilms, with a unique pattern that was dependent on temperature, time in culture, and position within the maturing biofilm. Tfp expression was required for the formation of the characteristic tower structures of NTHI biofilms and was significantly upregulated in NTHI biofilms formed at 34°C versus 37°C. This increase correlated with significantly greater twitching motility at 34°C than at 37°C. Treatment with antisera targeting the major subunit of Tfp (PilA) significantly inhibited NTHI biofilm formation at both temperatures, confirming the importance of this critical adhesin in biofilm formation. Additionally, treatment of preestablished biofilms with antisera against PilA significantly decreased biofilm biomass and mean thickness at both temperatures. These results demonstrated a pivotal role for Tfp in NTHI biofilm formation and stability at the temperature of the hNP, and they underscore the utility of PilA as a vaccine candidate for treatment and/or prevention of NTHI biofilm-associated diseases. IMPORTANCE: NTHI is an important cause of chronic respiratory tract infections, including otitis media, chronic rhinosinusitis, and exacerbations of chronic obstructive pulmonary disease and cystic fibrosis. The chronic and recurrent nature of these diseases is attributed to the presence of bacterial biofilms, which are highly resistant to antimicrobials. We characterized NTHI biofilm growth and expression of PilA, the major subunit of the Tfp, at the temperature of the hNP, which is the commensal habitat of NTHI. Our results expand the current understanding of the role of Tfp during biofilm formation and maturation at the temperature of both the hNP and the middle ear, and they strengthen support for PilA as a vaccine candidate for the prevention and treatment of NTHI biofilm-associated diseases.

DNABII proteins play a central role in UPEC biofilm structure.

Most chronic and recurrent bacterial infections involve a biofilm component, the foundation of which is the extracellular polymeric substance (EPS). Extracellular DNA (eDNA) is a conserved and key component of the EPS of pathogenic biofilms. The DNABII protein family includes integration host factor (IHF) and histone-like protein (HU); both are present in the extracellular milieu. We have shown previously that the DNABII proteins are often found in association with eDNA and are critical for the structural integrity of bacterial communities that utilize eDNA as a matrix component. Here, we demonstrate that uropathogenic Escherichia coli (UPEC) strain UTI89 incorporates eDNA within its biofilm matrix and that the DNABII proteins are not only important for biofilm growth, but are limiting; exogenous addition of these proteins promotes biofilm formation that is dependent on eDNA. In addition, we show that both subunits of IHF, yet only one subunit of HU (HupB), are critical for UPEC biofilm development. We discuss the roles of these proteins in context of the UPEC EPS.

Antibodies against the majority subunit of type IV Pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS-dependent manner and confer therapeutic resolution of experimental otitis media.

Despite resulting in a similar overall outcome, unlike antibodies directed against the DNABII protein, integration host factor (IHF), which induce catastrophic structural collapse of biofilms formed by nontypeable Haemophilus influenzae (NTHI), those directed against a recombinant soluble form of PilA [the majority subunit of Type IV pili (Tfp) produced by NTHI], mediated gradual 'top-down' dispersal of NTHI from biofilms. This dispersal occurred via a mechanism that was dependent upon expression of both PilA (and by inference, Tfp) and production of AI-2 quorum signaling molecules by LuxS. The addition of rsPilA to a biofilm-targeted therapeutic vaccine formulation comprised of IHF plus the powerful adjuvant dmLT and delivered via a noninvasive transcutaneous immunization route induced an immune response that targeted two important determinants essential for biofilm formation by NTHI. This resulted in significantly earlier eradication of NTHI from both planktonic and adherent populations in the middle ear, disruption of mucosal biofilms already resident within middle ears prior to immunization and rapid resolution of signs of disease in an animal model of experimental otitis media. These data support continued development of this novel combinatorial immunization approach for resolution and/or prevention of multiple diseases of the respiratory tract caused by NTHI.

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens.

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A ß-(1→6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.

Selection and Counterselection of Hia Expression Reveals a Key Role for Phase-Variable Expression of Hia in Infection Caused by Nontypeable Haemophilus influenzae.

Hia is a major adhesin of nontypeable Haemophilus influenzae (NTHi) and has long been investigated as a vaccine candidate. Here we show that Hia phase variation is controlled by changes in the length of a polythymidine tract located in the hia promoter. Studies of an invasive clinical isolate (strain R2866) show that strains expressing high Hia levels are more efficiently killed by opsonophagocytosis. An opsonophagocytic assay was used to select for a subpopulation of variants that expressed a low level of Hia, which facilitated their escape from killing by anti-Hia antisera. Conversely, a subpopulation of variants expressing a high level of Hia was selected for during passaging through Chang cells. In both cases, phase variation of Hia expression corresponded directly with discrete modal changes in polythymidine tract length. In the chinchilla model of NTHi infection, we observed consistent selection for high Hia expression upon nasopharyngeal colonization, confirming the key role of phase-variable expression of Hia within a specific niche in vivo.