Regulation of virulence gene expression resulting from Streptococcus pneumoniae and nontypeable Haemophilus influenzae interactions in chronic disease

Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and coculture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR) was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS

Sub-inhibitory fosmidomycin exposures elicits oxidative stress in Salmonella enterica Serovar typhimurium LT2

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RTPCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle)

Microbial interactions and differential protein expression in Staphylococcus aureus –Candida albicans dual-species biofilms

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection

Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms

Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm

Murine Immune Response to a Chronic Staphylococcus aureus Biofilm Infection ▿ †

Staphylococcus aureus has reemerged as an important human pathogen in recent decades. Although many infections caused by this microbial species persist through a biofilm mode of growth, little is known about how the host's adaptive immune system responds to these biofilm infections. In this study, S. aureus cells adhered to pins in culture and were subsequently inserted into the tibiae of C57BL/6 mice, with an infecting dose of 2 × 105 CFU. This model was utilized to determine local cytokine levels, antibody (Ab) function, and T cell populations at multiple time points throughout infection. Like human hosts, S. aureus implant infection was chronic and remained localized in 100% of C57BL/6 mice at a consistent level of approximately 107 CFU/gram bone tissue after day 7. This infection persisted locally for >49 days and was recalcitrant to clearance by the host immune response and antimicrobial therapy. Local inflammatory cytokines of the Th1 (interleukin-2 [IL-2], IL-12 p70, tumor necrosis factor alpha [TNF-α], and IL-1β) and Th17 (IL-6 and IL-17) responses were upregulated throughout the infection, except IL-12 p70, which dwindled late in the infection. In addition, Th1 Ab subtypes against a biofilm antigen (SA0486) were upregulated early in the infection, while Th2 Abs and anti-inflammatory regulatory T cells (Tregs) were not upregulated until later. These results indicate that early Th1 and Th17 inflammatory responses and downregulated Th2 and Treg responses occur during the development of a chronic biofilm implant infection. This unrestrained inflammatory response may cause tissue damage, thereby enabling S. aureus to attach and thrive in a biofilm mode of growth

Immunoglobulins to Surface-Associated Biofilm Immunogens Provide a Novel Means of Visualization of Methicillin-Resistant Staphylococcus aureus Biofilms▿

Antigens from the methicillin-resistant Staphylococcus aureus (MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships

Resolution of Staphylococcus aureus Biofilm Infection Using Vaccination and Antibiotic Treatment ▿ †

Staphylococcus aureus infections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, both in vitro and in vivo. Antibodies against these antigens were first used in microscopy studies to localize their expression in in vitro biofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups untreated or treated with vancomycin. Lastly, MRSA biofilm infections were significantly cleared in 87.5% of vaccinated and antibiotic-treated animals, while antibiotics or vaccine alone could not significantly clear infection compared to controls (55.6, 22.2, and 33.3% clearance rates, respectively). This approach to vaccine development may lead to the generation of vaccines against other pathogenic biofilm bacteria