Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

Dinner date: Neisseria gonorrhoeae central carbon metabolism and pathogenesis.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is a human-adapted pathogen that does not productively infect other organisms. The ongoing relationship between N. gonorrhoeae and the human host is facilitated by the exchange of nutrient resources that allow for N. gonorrhoeae growth in the human genital tract. What N. gonorrhoeae 'eats' and the pathways used to consume these nutrients have been a topic of investigation over the last 50 years. More recent investigations are uncovering the impact of N. gonorrhoeae metabolism on infection and inflammatory responses, the environmental influences driving N. gonorrhoeae metabolism, and the metabolic adaptations enabling antimicrobial resistance. This mini-review is an introduction to the field of N. gonorrhoeae central carbon metabolism in the context of pathogenesis. It summarizes the foundational work used to characterize N. gonorrhoeae central metabolic pathways and the effects of these pathways on disease outcomes, and highlights some of the most recent advances and themes under current investigation. This review ends with a brief description of the current outlook and technologies under development to increase understanding of how the pathogenic potential of N. gonorrhoeae is enabled by metabolic adaptation.

Transcriptome-guided metabolic network analysis reveals rearrangements of carbon flux distribution in Neisseria gonorrhoeae during neutrophil co-culture.

The ability of bacterial pathogens to metabolically adapt to the environmental conditions of their hosts is critical to both colonization and invasive disease. Infection with Neisseria gonorrhoeae (the gonococcus, Gc) is characterized by the influx of neutrophils [polymorphonuclear leukocytes (PMNs)], which fail to clear the bacteria and make antimicrobial products that can exacerbate tissue damage. The inability of the human host to clear Gc infection is particularly concerning in light of the emergence of strains that are resistant to all clinically recommended antibiotics. Bacterial metabolism represents a promising target for the development of new therapeutics against Gc. Here, we generated a curated genome-scale metabolic network reconstruction (GENRE) of Gc strain FA1090. This GENRE links genetic information to metabolic phenotypes and predicts Gc biomass synthesis and energy consumption. We validated this model with published data and in new results reported here. Contextualization of this model using the transcriptional profile of Gc exposed to PMNs revealed substantial rearrangements of Gc central metabolism and induction of Gc nutrient acquisition strategies for alternate carbon source use. These features enhanced the growth of Gc in the presence of neutrophils. From these results, we conclude that the metabolic interplay between Gc and PMNs helps define infection outcomes. The use of transcriptional profiling and metabolic modeling to reveal new mechanisms by which Gc persists in the presence of PMNs uncovers unique aspects of metabolism in this fastidious bacterium, which could be targeted to block infection and thereby reduce the burden of gonorrhea in the human population. IMPORTANCE The World Health Organization designated Gc as a high-priority pathogen for research and development of new antimicrobials. Bacterial metabolism is a promising target for new antimicrobials, as metabolic enzymes are widely conserved among bacterial strains and are critical for nutrient acquisition and survival within the human host. Here we used genome-scale metabolic modeling to characterize the core metabolic pathways of this fastidious bacterium and to uncover the pathways used by Gc during culture with primary human immune cells. These analyses revealed that Gc relies on different metabolic pathways during co-culture with human neutrophils than in rich media. Conditionally essential genes emerging from these analyses were validated experimentally. These results show that metabolic adaptation in the context of innate immunity is important to Gc pathogenesis. Identifying the metabolic pathways used by Gc during infection can highlight new therapeutic targets for drug-resistant gonorrhea.

Improving the identification of patients with a genetic diagnosis of familial hypercholesterolaemia in primary care: A strategy to achieve the NHS long term plan.

BACKGROUND AND AIMS: We aimed to validate a nurse-led process using electronic health records to identify those at risk of familial hypercholesterolaemia (FH) for genetic diagnosis in primary care. METHODS: Those at risk of FH were identified using searches developed and refined locally and implemented in primary care by a trained nurse; they were invited for further assessment and genetic testing if indicated. Family members at risk of FH were identified and invited for cascade testing. RESULTS: In total 94,444 patient records were screened (expected prevalence of FH (1 in 250); 377). Of 176 records which already had a diagnostic for FH, 15 had been genetically confirmed and one was undergoing DNA testing. A further 572 (0.61%) were identified as high risk of FH. After desktop screening, 113 (15%) were invited for further assessment. Of these, 73 individuals attended the primary care clinic (64%) of whom 61 (54%) underwent proband genetic testing. Pathogenic variants were detected in 22 cases (36%) and variants of unknown significance in a further 4 cases; a total of 26 probands (43%) were therefore referred for family cascade testing. CONCLUSIONS: An optimised FH identification pathway, based on the NICE CG71 recommendations for systematic searching of primary care electronic health records, can be deployed successfully in primary care settings.

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection.

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.