Lawrence K. Silbart

Outer Membrane Protein A (OmpA) of Cronobacter sakazakii Binds Fibronectin and Contributes to Invasion of Human Brain Microvascular Endothelial Cells

Cronobacter sakazakii is an emerging foodborne pathogen that causes severe meningitis and meningoencephalitis in neonates. Currently there is a dearth of information available on the virulence factors of C. sakazakii and the pathogenic mechanisms involved in its neonatal infections. The invasion and translocation of the blood-brain barrier formed by brain microvascular endothelial cells (BMEC) is critical in the pathogenesis of neonatal bacterial meningitis. Because bacterial binding of fibronectin is an initial step in the invasion of BMEC, the role of a major surface-expressed fibronectin-binding protein of C. sakazakii in invasion of BMEC was investigated. Outer membrane protein A was identified as a major fibronectin-binding protein of C. sakazakii, and an isogenic ompA mutant of C. sakazakii exhibited significantly (p < 0.05) attenuated invasion in BMEC compared with the wild-type strain. The findings of this study indicate that outer membrane protein A is one of the determinants that contribute to C. sakazakii invasion of human BMEC in vitro, and may potentially play a role in the pathogenesis of neonatal meningitis caused by this organism.

Correlates of Immune Protection in Chickens Vaccinated with Mycoplasma gallisepticum Strain GT5 following Challenge with Pathogenic M. gallisepticum Strain Rlow

ABSTRACT Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain Rlow. GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4+, and CD8+ cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with Rlow. Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4+ and CD8+ cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease

The aim of this study was to assess the efficacy of a modified live Mycoplasma gallisepticum vaccine (GT5) for the protection of chickens against infection and respiratory disease. GT5 was constructed by the reconstitution of the avirulent high passage R (R(high)) strain with the gene encoding the major cytadhesin GapA. GT5 expressed GapA on its surface yet retained the phenotypic characteristics of the parental R(high) strain. Birds vaccinated with GT5 were protected upon challenge with the virulent low passage R (R(low)) strain as evidenced by a complete absence of tracheal lesions 2 and 4 weeks post-challenge, in contrast to sham immunized/challenged control birds. Modest amounts of IgG, and little, if any secretory IgA or IgM anti-M. gallisepticum were found in tracheal washings following vaccination. However, copious amounts of specific IgA were found following challenge, especially in sham immunized birds. This suggests that the tracheal IgG elicited by GT5 vaccination may have been responsible for blocking the initial colonization of R(low), thereby resulting in protection.

A Microarray Biosensor for Multiplexed Detection of Microbes Using Grating-Coupled Surface Plasmon Resonance Imaging

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a nontoxic form of Pseudomonas aeruginosa exotoxin A (ntPE), Bacillus globigii, Mycoplasma hyopneumoniae, Listeria monocytogenes, Escherichia coli, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free, and highly multiplexed assay requiring nanoliter amounts of capture reagents.

Comparative assessment of a metabolically attenuated Mycoplasma gallisepticum mutant as a live vaccine for the prevention of avian respiratory mycoplasmosis.

In a previous study, signature sequence mutagenesis (SSM) was used to identify a mutant with a disruption of the gene encoding the metabolic factor, dihydrolipoamide dehydrogenase, and that mutant was designated Mg 7. The current study assessed the safety, immunogenicity and efficacy of Mg 7 in comparison to two commercially available vaccines (ts-11 and F) as well as a laboratory vaccine strain, GT5. Intratracheal vaccination of chickens with all four attenuated mutants induced varying levels of protection against intratracheal challenge with virulent Mycoplasma gallisepticum strain R(low). Mg 7 vaccinated chickens rapidly cleared the challenge strain, had lower histopathologic tracheal lesion scores when compared to unvaccinated chickens, and mounted a strong humoral anti-M. gallisepticum-specific IgG response. The IgG levels increased 2- to 3-fold upon R(low) challenge. Mg 7 induced a greater level of protection against intratracheal R(low) challenge than that observed with the other three attenuated strains, as evidenced by a lower recovery of R(low) from tracheas and lower histopathologic lesion scores in tracheas and air sacs. Based on these findings, Mg 7 appears to have good potential as a safe and effective vaccine for the prevention of avian mycoplasmosis.

Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

ABSTRACT Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

Broncho-alveolar macrophages express chemokines associated with leukocyte migration in a mouse model of asthma

The migration of eosinophils and lymphocytes into airways is a hallmark of allergic asthma; however, the role of broncho-alveolar macrophages (BAMs) in this inflammatory process has not been fully elucidated. Using a murine Ova model of allergic airway disease (AAD), RNA isolated from BAMs was used to assess differential gene expression via microarray and qRT-PCR. Significant increases in WBCs, eosinophilia, mucus accumulation and goblet cell hyperplasia were observed in Ova sensitized and challenged mice, which correlated with increased expression of genes associated with alternatively activated M2 macrophages (e.g. arginase 1, YM-1, YM-2, Resistin like-α, and EAR-11). Other genes associated with asthma including FcγRIIb, MMP-14, CCL-8, CCL-17, ADAM-8, LTBR1, aquaporin-9 and IL-7R were also expressed at higher levels in Ova sensitized/challenged animals when compared to BAMs isolated from control animals. Eotaxin 2 (CCL-24), which is known to influence eosinophil migration, was highly up-regulated in BAMs, but not Eotaxin-1 (CCL-11). Conversely, lung interstitial macrophages expressed high levels of CCL-11, but not CCL-24. Taken together, this study provides additional evidence to support the notion that M2 BAMs play a role in eosinophil and potentially other leukocyte migration patterns into asthmatic airways.

Pulmonary eosinophilia correlates with allergen deposition to the lower respiratory tract in a mouse model of asthma

Background Eosinophilic infiltration into the airways is frequently associated with allergic asthma; however, the role of antigen deposition in mediating this phenomenon has not been studied in detail.

Mycoplasma gallisepticum lipid associated membrane proteins up-regulate inflammatory genes in chicken tracheal epithelial cells via TLR-2 ligation through an NF-κB dependent pathway.

Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (Rlow) or a non-virulent (Rhigh) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, Rlow exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2–3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either Rlow or Rhigh exposure. Taken together we conclude that LAMPs isolated from both Rhigh and Rlow induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway.