Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways

Abstract

Antibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010–2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways

“…A genome-wide screen for H. influenzae genes required in the lung previously revealed vacJ involvement in pathogenesis 18 . Following this observation, we sought to quantify the impact of vacJ disruption in a NTHi mouse respiratory infection model system previously used for NTHi375 29 , 31 – 34 . Mice were infected with NTHi375 WT and vacJ mutant strains, and bacterial loads quantified in lung and bronchoalveolar lavage fluid (BALF) samples at 24 and 48 h post-infection (hpi).…”

Moonlighting of Haemophilus influenzae heme acquisition systems contributes to the host airway-pathogen interplay in a coordinated manner

“…S7B). Last, we used an in vivo mouse respiratory infection model system [79][80][81]85,86]. At 24 hpi, NTHi375ΔhxuCBA lung and BALF bacterial numbers were lower than those recovered for the WT strain ( Figure 6(c), left panels).…”

“…A549 type II human pneumocytes (ATCC CCL-185) were maintained and seeded as described [79][80][81][82][83][84][85][86]. For infection, PBS-normalized bacterial suspensions (OD 600 = 1) were prepared by using NTHi strains grown on chocolate agar.…”

“…A mouse model of NTHi lung infection was used as described previously [86]. CD1 female mice were purchased from Charles River Laboratories (France), housed under pathogen-free conditions at the Institute of Agrobiotechnology facilities (registration number 365 ES/31-2016-000002-CR-SU-US), and used at 22-25 g (6-7 weeks).…”

Preclinical Evaluation of the Antimicrobial-Immunomodulatory Dual Action of Xenohormetic Molecules against Haemophilus influenzae Respiratory Infection

“…Based on these observations, quercetin was shortlisted to next assess its modulatory effect on the NTHi-host airway interplay. NTHi375 was used for this purpose, given that it has been previously used in host-pathogen interaction studies [42,[47][48][49].…”