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S-nitrosylation is an important mediator of multiple nitric oxide-dependent biological processes, including eukaryotic cellular events such as macrophage apoptosis and proinflammatory signaling. Many pathogenic bacteria possess NO detoxification mechanisms, such as the nitric oxide reductase (NorB) of Neisseria meningitidis and the flavohemoglobins (Hmp) of Salmonella enterica and Escherichia coli, which serve to protect the microorganism from nitrosative stress within the intracellular environment. In this study, we demonstrate that expression of meningococcal NorB increases the rate at which low-molecular-weight S-nitrosothiol (SNO) decomposes in vitro. To determine whether this effect occurs in cells during infection by bacteria, we induced SNO formation in murine macrophages by activation with lipopolysaccharide and γ-interferon and observed a reduced abundance of SNO during coincubation with N. meningitidis, S. enterica, or E. coli. In each case, this effect was shown to be dependent on bacterial NO detoxification genes, which act to prevent SNO formation through the removal of NO. This may represent a novel mechanism of host cell injury by bacteria.—Laver, J. R., Stevanin, T. M., Messenger, S. L., Dehn Lunn, A., Lee, M. E., Moir, J. W. B., Poole, R. K., Read, R. C. Bacterial nitric oxide detoxification prevents host cell S-nitrosothiol formation: a novel mechanism of bacterial pathogenesis.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution.

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IntroductionInfant upper respiratory microbiota are derived partly from the maternal respiratory tract, and certain microbiota are associated with altered risk of infections and respiratory disease. Neisseria lactamica is a common pharyngeal commensal in young children and is associated with reduced carriage and invasive disease by Neisseria meningitidis. Nasal inoculation with N. lactamica safely and reproducibly reduces N. meningitidis colonisation in healthy adults. We propose nasal inoculation of pregnant women with N. lactamica, to establish if neonatal pharyngeal colonisation occurs after birth, and to characterise microbiome evolution in mother–infant pairs over 1 month post partum.Methods and analysis20 healthy pregnant women will receive nasal inoculation with N. lactamica (wild type strain Y92-1009) at 36–38 weeks gestation. Upper respiratory samples, as well as optional breastmilk, umbilical cord blood and infant venous blood samples, will be collected from mother–infant pairs over 1 month post partum. We will assess safety, N. lactamica colonisation (by targeted PCR) and longitudinal microevolution (by whole genome sequencing), and microbiome evolution (by 16S rRNA gene sequencing).Ethics and disseminationThis study has been approved by the London Central Research Ethics Committee (21/PR/0373). Findings will be published in peer-reviewed open-access journals as soon as possible.Trial registration numberNCT04784845.

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Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that plays a major role in a number of respiratory tract infections, including otitis media, cystic fibrosis and chronic obstructive pulmonary disease. Biofilm formation has been implicated in both NTHi colonization and disease, and is responsible for the increased tolerance of this pathogen towards antibiotic treatment. Targeting metabolic pathways that are important in NTHi biofilm formation represents a potential strategy to combat this antibiotic recalcitrance. A previous investigation demonstrated increased expression of a putative d-methionine uptake protein following exposure of NTHi biofilms to the ubiquitous signalling molecule, nitric oxide. We therefore hypothesized that treatment with exogenous d-methionine would impact on NTHi biofilm formation and increase antibiotic sensitivity. Treatment of NTHi during the process of biofilm formation resulted in a reduction in biofilm viability, increased biomass, changes in the overall biofilm architecture and the adoption of an amorphous cellular morphology. Quantitative proteomic analyses identified 124 proteins that were differentially expressed following d-methionine treatment, of which 51 (41 %) were involved in metabolic and transport processes. Nine proteins involved in peptidoglycan synthesis and cell division showed significantly increased expression. Furthermore, d-methionine treatment augmented the efficacy of azithromycin treatment and highlighted the potential of d-methionine as an adjunctive therapeutic approach for NTHi biofilm-associated infections.