Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae

We have developed three nontypeable Haemophilus influenzae (NTHI) adhesin-derived immunogens that are significantly efficacious against experimental otitis media (OM) due to NTHI when delivered parenterally. We now expanded our preventative immunization strategies to include transcutaneous immunization (TCI) as a less invasive, but potentially equally efficacious, regimen to prevent OM due to NTHI. Additionally, we examined the potential of TCI as a therapeutic immunization regimen to resolve ongoing experimental OM. Preventative immunization with NTHI outer membrane protein (OMP) P5- and type IV pilus-targeted immunogens, delivered with the adjuvant LT(R192G-L211A), induced significantly earlier clearance of NTHI from the nasopharynges and middle ears of challenged chinchillas compared with receipt of immunogen or adjuvant alone. Moreover, therapeutic immunization resulted in significant resolution of established NTHI biofilms from the middle ear space of animals compared with controls. These data advocate TCI with the adhesin-directed immunogens as an efficacious regimen for prevention and resolution of experimental NTHI-induced OM

The Multifunctional Host Defense Peptide SPLUNC1 Is Critical for Homeostasis of the Mammalian Upper Airway

Otitis media (OM) is a highly prevalent pediatric disease caused by normal flora of the nasopharynx that ascend the Eustachian tube and enter the middle ear. As OM is a disease of opportunity, it is critical to gain an increased understanding of immune system components that are operational in the upper airway and aid in prevention of this disease. SPLUNC1 is an antimicrobial host defense peptide that is hypothesized to contribute to the health of the airway both through bactericidal and non-bactericidal mechanisms. We used small interfering RNA (siRNA) technology to knock down expression of the chinchilla ortholog of human SPLUNC1 (cSPLUNC1) to begin to determine the role that this protein played in prevention of OM. We showed that knock down of cSPLUNC1 expression did not impact survival of nontypeable Haemophilus influenzae, a predominant causative agent of OM, in the chinchilla middle ear under the conditions tested. In contrast, expression of cSPLUNC1 was essential for maintenance of middle ear pressure and efficient mucociliary clearance, key defense mechanisms of the tubotympanum. Collectively, our data have provided the first in vivo evidence that cSPLUNC1 functions to maintain homeostasis of the upper airway and, thereby, is critical for protection of the middle ear

Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology

Background Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi. Results A cassette containing a spectinomycin resistance gene and an rpsL gene flanked by FRT sites was constructed. A PCR amplicon containing 50 base pairs of DNA homologous to the 5' and 3' ends of the gene to be disrupted and the cassette was generated, then recombineered into the target NTHi gene, cloned on a plasmid, using the lambda recombination proteins expressed in E. coli DY380. Thus, the gene of interest was replaced by the cassette. The construct was then transformed into a streptomycin resistant NTHi strain and mutants were selected on spectinomycin-containing growth media. A plasmid derived from pLS88 with a temperature sensitive replicon expressing the FLP recombinase gene under the control of the tet operator/repressor was constructed. This plasmid was electroporated into the NTHi mutant at the permissive temperature and FLP expression was induced using anhydrotetracycline. The recombinase recognizes the FRT sites and eliminates the antibiotic cassette by site-specific recombination, creating the unmarked non-polar mutation. The plasmid is cured by growth of cells at the restrictive temperature. Conclusion The products of the genes in the NTHi pilABCD operon are required for type IV pilus biogenesis and have a role in transformation. We demonstrated the utility of our methodology by the construction of a non-polar pilA mutation in NTHi strain 2019 and complementation of the mutation with a plasmid containing the pilA gene. Utilization of this approach allowed us to readily generate unmarked non-polar mutations in NTHi genes.This work was supported by NIH grants R01DC007464 to RSM, R01DC003915 to Lauren Bakaletz and a subcontract from N01AI30040 to Michael Apicella. We thank Michael Apicella for the gifts of NTHi strains 2019 and 2019 rpsL

The central region of the msp gene of Treponema denticola has sequence heterogeneity among clinical samples, obtained from patients with periodontitis

<p>Abstract</p> <p>Background</p> <p><it>Treponema denticola </it>is an oral spirochete involved in the pathogenesis and progression of periodontal disease. Of its virulence factors, the major surface protein (MSP) plays a role in the interaction between the treponeme and host. To understand the possible evolution of this protein, we analyzed the sequence of the <it>msp </it>gene in 17 <it>T. denticola </it>positive clinical samples.</p> <p>Methods</p> <p>Nucleotide and amino acid sequence of MSP have been determined by PCR amplification and sequencing in seventeen <it>T. denticola </it>clinical specimens to evaluate the genetic variability and the philogenetic relationship of the <it>T. denticola msp </it>gene among the different amplified sequence of positive samples. In silico antigenic analysis was performed on each MSP sequences to determined possible antigenic variation.</p> <p>Results</p> <p>The <it>msp </it>sequences showed two highly conserved 5' and 3' ends and a central region that varies substantially. Phylogenetic analysis categorized the 17 specimens into 2 principal groups, suggesting a low rate of evolutionary variability and an elevated degree of conservation of <it>msp </it>in clinically derived genetic material. Analysis of the predicted antigenic variability between isolates, demonstrated that the major differences lay between amino acids 200 and 300.</p> <p>Conclusion</p> <p>These findings showed for the first time, the nucleotide and amino acids variation of the <it>msp </it>gene in infecting <it>T. denticola</it>, <it>in vivo</it>. This data suggested that the antigenic variability found in to the MSP molecule, may be an important factor involved in immune evasion by <it>T. denticola</it>.</p

Viral-bacterial co-infection in Australian Indigenous children with acute otitis media

Background: Acute otitis media with perforation (AOMwiP) affects 40% of remote Indigenous children during the first 18 months of life. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are the primary bacterial pathogens of otitis media and their loads predict clinical ear state. Our hypothesis is that antecedent respiratory viral infection increases bacterial density and progression to perforation

High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

Secondary bacterial infection in humans is one of the pathological conditions requiring clinical attention. In this study, we examined the effect of lipopolysaccharide (LPS) on encephalomyocarditis virus (EMCV) infected mice. All mice inoculated with EMCV at 5 days before LPS challenge died within 24 h. LPS-induced TNF-α mRNA expression was significantly increased in the brain and heart at 5 days after EMCV infection. CD11b+/TLR4+ cell population in the heart was remarkably elevated at 5 days after EMCV infection, and sorted CD11b+ cells at 5 days after EMCV infection produced a large amount of TNF-α on LPS stimulation in vivo and in vitro. In conclusion, we found that the infiltration of CD11b+ cells into infected organs is involved in the subsequent LPS-induced lethal shock in viral encephalomyocarditis. This new experimental model can help define the mechanism by which secondary bacterial infection causes a lethal shock in viral encephalomyocarditis

CNS Recruitment of CD8+ T Lymphocytes Specific for a Peripheral Virus Infection Triggers Neuropathogenesis during Polymicrobial Challenge

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ∼50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack

Quantitative PCR of ear discharge from Indigenous Australian children with acute otitis media with perforation supports a role for Alloiococcus otitidis as a secondary pathogen

Otitis media is endemic in remote Indigenous communities of Australia’s Northern Territory. Alloiococcus otitidis is an outer ear commensal and putative middle ear pathogen that has not previously been described in acute otitis media (AOM) in this population. The aims of this study were to determine the presence, antibiotic susceptibility and bacterial load of A. otitidis in nasopharyngeal and ear discharge swabs collected from Indigenous Australian children with AOM with perforation.Financial support for this study was provided by the Channel 7 Children’s Research Foundation; The Trust Foundation; and the National Health and Medical Research Council (Australia)

Metabolite Cross-Feeding Enhances Virulence in a Model Polymicrobial Infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities

A three-way comparative genomic analysis of Mannheimia haemolytica isolates

<p>Abstract</p> <p>Background</p> <p><it>Mannhemia haemolytica </it>is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of <it>M. haemolytica </it>at molecular level and assist in devising an effective control strategy.</p> <p>Description</p> <p>During our comparative genomic sequence analysis of three <it>Mannheimia haemolytica </it>isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors.</p> <p>Conclusions</p> <p>During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in <it>M. haemolytica </it>will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.</p