Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

Abstract

A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease. Molecular & Cellular Proteomics 15: 10.1074/mcp.M115.052498, 1117-1138, 2016.Our current understanding of the global changes that occur during infection is primarily based upon transcriptional profiles of either the host or the bacteria. However, a significant component of disease progression is dependent upon posttranscriptional processes. Recent advances in the application of two-dimensional tandem mass spectrometry have dramatically increased sensitivity to allow simultaneous analyses of multiple molecules from very small biological samples. In this study, we report the comprehensive proteomic and metabolomic profiling of middle ear tissues using samples that are smaller than those typically obtained from a human biopsy. Proteomic and metabolomic analyses of samples as small as biopsies will revolutionize the elucidation of the mechanisms From the

Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

“…7 ). Similarly, we have previously shown that inhibition of the actin-related protein complex Arp2/3 prevents NTHI uptake into epithelial cells ( 70 ). Therefore, it is tempting to speculate that the use of macropinocytosis or actin inhibitors could prevent NTHI persistence and reduce recurrent episodes of OM.…”

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence

“…This model provides a new platform for the identification of novel diagnostic and therapeutic approaches for OM. Our prior proteomic studies demonstrate an increase in the actin filament branching protein, Arp2/3, during acute experimental OM and that inhibition of Arp2/3 decreases NTHI invasion as a potential adjunct therapy [ 64 ]. Finally, it is of interest to determine whether intracellular populations contribute to the other NTHI-mediated diseases such as conjunctivitis, sinusitis, pneumonia, and exacerbations in COPD and cystic fibrosis [ 65 , 66 ].…”

“…On days 7, 14, and 28, chinchillas were sacrificed and the middle ears removed, fixed in 4% paraformaldehyde in Dulbecco’s Phosphate Buffered Saline (DPBS), decalcified in 0.35 M Tris/EDTA solution and embedded in paraffin. Thin sections from fixed middle ears were deparaffinized in xylene and antigen-retrieval was performed as previously described [ 64 ] followed by treatment with 0.01% sodium borohydride (Thermo Fisher Scientific) in DPBS for 10 minutes to inhibit inherent fluorescence due to aldehyde production. Slides were then incubated for 10 minutes in CAS Block (Thermo Fisher Scientific) to prevent non-specific staining followed by a 30-minute incubation with Image-iT FX Signal Enhancer (Thermo Fisher Scientific).…”

Continuous Microevolution Accelerates Disease Progression during Sequential Episodes of Infection

“…We previously demonstrated that nutritional sequestration influences disease progression and severity, microevolution of NTHI during stationary phase, and establishment of an intracellular NTHI population Szelestey et al, 2013). Host microenvironmental influences on NTHI pathoadaptation and evolution have revealed additional targets for treatment of OM based upon the multiple pathogenic lifestyles revealed in our studies (e.g., biofilm, intracellular) Harrison et al, 2016;Szelestey et al, 2013). In order to evaluate the effect of multiple episodes of OM on the microevolution of NTHI, we established a model system of sequential OM episodes through naive hosts.…”