Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Role of Phenol-Soluble Modulins in Formation of Staphylococcus aureus Biofilms in Synovial Fluid.

Staphylococcus aureus is a leading cause of prosthetic joint infections, which, as we recently showed, proceed with the involvement of biofilm-like clusters that cause recalcitrance to antibiotic treatment. Here we analyzed why these clusters grow extraordinarily large, reaching macroscopically visible extensions (\u3e1 mm). We found that while specific S. aureus surface proteins are a prerequisite for agglomeration in synovial fluid, low activity of the Agr regulatory system and subsequent low production of the phenol-soluble modulin (PSM) surfactant peptides cause agglomerates to grow to exceptional dimensions. Our results indicate that PSMs function by disrupting interactions of biofilm matrix molecules, such as the polysaccharide intercellular adhesin (PIA), with the bacterial cell surface. Together, our findings support a two-step model of staphylococcal prosthetic joint infection: As we previously reported, interaction of S. aureus surface proteins with host matrix proteins such as fibrin initiates agglomeration; our present results show that, thereafter, the bacterial agglomerates grow to extremely large sizes owing to the lack of PSM expression under the specific conditions present in joints. Our findings provide a mechanistic explanation for the reported extreme resistance of joint infection to antibiotic treatment, lend support to the notions that Agr functionality and PSM production play a major role in defining different forms of S. aureus infection, and have important implications for antistaphylococcal therapeutic strategies

Toxin Mediates Sepsis Caused by Methicillin-Resistant <i>Staphylococcus epidermidis</i>

<div><p>Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species <i>Staphylococcus epidermidis</i> are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant <i>S</i>. <i>epidermidis</i> is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the <i>psm-mec</i> locus that have been reported in <i>S</i>. <i>aureus</i> strains, inasmuch as our findings suggest that the <i>psm-mec</i> locus may exert effects in the background of <i>S</i>. <i>aureus</i> strains that differ from its original role in the CNS environment due to originally “unintended” interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.</p></div

PSM production in <i>S</i>. <i>epidermidis</i> strains and isogenic <i>psm-mec</i> deletion and <i>psm-mec</i> start codon mutants.

<p>PSMs were measured in stationary phase (16 h) cultures using RP-HPLC/ESI-MS in triplicate. Error bars show ±SEM. $, Residual PSM-mec production in the SE620 <i>psm-mec</i> start codon mutant is likely due to strong gene expression and usage of a non-canonical start codon, as we previously found also in <i>S</i>. <i>aureus psm-mec</i> start codon mutants [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006153#ppat.1006153.ref014" target="_blank">14</a>].</p

Bacterial survival during incubation with human neutrophils and in whole human blood.

<p>(<b>A,B</b>) Survival in whole, heparinized human blood. (<b>C,D</b>) Survival during incubation with human neutrophils (MOI 10:1). (<b>E,F</b>) Killing of human neutrophils (MOI 100:1). Error bars show ±SEM. *, <i>P</i> <0.05, **, <i>P</i> <0.01. ***, <i>P</i> <0.001. ****, <i>P</i> <0.0001 (unpaired t-tests for RP62A; 1-way ANOVA with Bonferroni post-tests for SE620). Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant. In (A) and (C), no comparisons between SE620Δ<i>psm-mec</i> and SE620<i>psm-mec</i>* were statistically significant.</p

Microarray results, RP62A¹.

<p>Microarray results, RP62A<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006153#t001fn001" target="_blank">¹</a>.</p

Time-dependent expression of <i>psm-mec</i>.

<p>Expression of the <i>psm-mec</i> RNA relative to that of 16S RNA in <i>S</i>. <i>epidermidis</i> RP62A during growth in TSB. The experiment was performed in triplicate. Error bars show ±SEM.</p

Mouse sepsis model.

<p>Female, 6–10 weeks old, C57BL/6NCRl mice (n = 5 for all groups except SE620Δ<i>psm-mec</i> and SE620<i>psm-mec</i>*, n = 10) were injected via the tail vein with 5 x 10⁸ CFU of the indicated bacterial strains and monitored for disease development every 8 h for up to 120 h. (<b>A,B</b>) Survival curves; (<b>C,D</b>) CFU in blood at 12 h; (<b>E,F</b>) CFU in kidneys at 12 h. Statistical analysis is by Log-rank (Mantel-Cox) tests for survival curves, otherwise using 1-way ANOVA with Bonferroni post-tests. Error bars show ±SEM. N.S., not significant. Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant.</p