Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro-and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont Haemophilus influenzae (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOClmediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of msrAB was also induced by hydrogen peroxide and paraquat, but a Hi2019 ΔmsrAB strain was not susceptible to killing by these ROS in vitro. Hi2019 ΔmsrAB fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019 ΔmsrAB led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019 WT , and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen in upper and lower respiratory tract infections in humans, and is increasingly also associated with invasive disease. We have examined two unrelated NTHi invasive disease isolates, R2866 and C188, in order to identify metabolic and physiological properties that distinguish them from respiratory tract disease isolates such as Hi2019. While the general use of the Hi metabolic network was similar across all three strains, the two invasive isolates secreted increased amounts of succinate, which can have anti-inflammatory properties. In addition, they showed a common shift in their carbon source utilization patterns, with strongly enhanced metabolism of nucleoside substrates, glucose and sialic acid. The latter two are major compounds present in blood and cerebrospinal fluid (CSF). Interestingly, C188 and R2866 also shared a reduced ability to invade or survive intracellularly in 16HBE14 bronchial epithelial cells relative to Hi2019 (4-fold (4 h), 25-fold (24 h) reduction). Altered metabolic properties, such as the ones observed here, could arise from genomic adaptations that NTHi undergo during infection. Together these data indicate that shifts in substrate preferences in otherwise conserved metabolic pathways may underlie strain niche specificity and thus have the potential to alter the outcomes of host–NTHi interactions.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo. Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease.

Haemophilus influenzae (Hi) infections are associated with recurring acute exacerbations of chronic respiratory diseases in children and adults including otitis media, pneumonia, chronic obstructive pulmonary disease and asthma. Here, we show that persistence and recurrence of Hi infections are closely linked to Hi metabolic properties, where preferred growth substrates are aligned to the metabolome of human airway epithelial surfaces and include lactate, pentoses, and nucleosides, but not glucose that is typically used for studies of Hi growth in vitro. Enzymatic and physiological investigations revealed that utilization of lactate, the preferred Hi carbon source, required the LldD L-lactate dehydrogenase (conservation: 98.8% of strains), but not the two redox-balancing D-lactate dehydrogenases Dld and LdhA. Utilization of preferred substrates was directly linked to Hi infection and persistence. When unable to utilize L-lactate or forced to rely on salvaged guanine, Hi showed reduced extra- and intra-cellular persistence in a murine model of lung infection and in primary normal human nasal epithelia, with up to 3000-fold attenuation observed in competitive infections. In contrast, D-lactate dehydrogenase mutants only showed a very slight reduction compared to the wild-type strain. Interestingly, acetate, the major Hi metabolic end-product, had anti-inflammatory effects on cultured human tissue cells in the presence of live but not heat-killed Hi, suggesting that metabolic endproducts also influence HI-host interactions. Our work provides significant new insights into the critical role of metabolism for Hi persistence in contact with host cells and reveals for the first time the immunomodulatory potential of Hi metabolites.

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.