Bridget Gollan

Temperature triggers immune evasion by Neisseria meningitidis

Neisseria meningitidis has several strategies to evade complement-mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature. We have identified three independent RNA thermosensors located in the 5′ untranslated regions of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing. Therefore increased temperature (which occurs during inflammation) acts as a ‘danger signal’ for the meningococcus, enhancing its defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an increased temperature and recruitment of immune effectors. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium.

A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies

The presence of serum bactericidal antibodies is a proven correlate of protection against systemic infection with the important human pathogen Neisseria meningitidis. We have identified three serogroup C N. meningitidis (MenC) isolates recovered from patients with invasive meningococcal disease that resist killing by bactericidal antibodies induced by the MenC conjugate vaccine. None of the patients had received the vaccine, which has been successfully introduced in countries in North America and Europe. The increased resistance was not caused by changes either in lipopolysaccharide sialylation or acetylation of the α2-9–linked polysialic acid capsule. Instead, the resistance of the isolates resulted from the presence of an insertion sequence, IS1301, in the intergenic region (IGR) between the sia and ctr operons, which are necessary for capsule biosynthesis and export, respectively. The insertion sequence led to an increase in the transcript levels of surrounding genes and the amount of capsule expressed by the strains. The increased amount of capsule was associated with down-regulation of the alternative pathway of complement activation, providing a generic mechanism by which the bacterium protects itself against bactericidal antibodies. The strains with IS1301 in the IGR avoided complement-mediated lysis in the presence of bactericidal antibodies directed at the outer membrane protein, PorA, or raised against whole cells.

Persistent bacterial infections and persister cells.

Many bacteria can infect and persist inside their hosts for long periods of time. This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics. Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug. Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress. The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change. The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment. If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections. In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections.

A Salmonella toxin promotes persister formation through acetylation of tRNA

Summary The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.

Toxin–antitoxin systems: reversible toxicity

Toxin-antitoxin (TA) systems encoded on the plasmids and chromosomes of bacteria are emerging as key players in stress adaptation. In particular, they have been implicated in the induction of persisters non-growing cells that can evade antibiotic exposure. TA toxins operate by a diverse range of mechanisms, either destructive or conservative, leading to the reversible growth arrest of bacterial cells. Whilst the molecular mechanisms of intoxication are now well understood, we still have very little information on how corrupted cells reawaken. Alongside the phenomenon of conditional cooperativity, new evidence suggests that the effects of some TA toxins can be reversed, allowing non-growing cells to be detoxified and growth to resume.

The Influence of IS1301 in the Capsule Biosynthesis Locus on Meningococcal Carriage and Disease

Previously we have shown that insertion of IS1301 in the sia/ctr intergenic region (IGR) of serogroup C Neisseria meningitidis (MenC) isolates from Spain confers increased resistance against complement-mediated killing. Here we investigate the significance of IS1301 in the same location in N. meningitidis isolates from the UK. PCR and sequencing was used to screen a collection of more than 1500 meningococcal carriage and disease isolates from the UK for the presence of IS1301 in the IGR. IS1301 was not identified in the IGR among vaccine failure strains but was frequently found in serogroup B isolates (MenB) from clonal complex 269 (cc269). Almost all IS1301 insertions in cc269 were associated with novel polymorphisms, and did not change capsule expression or resistance to human complement. After excluding sequence types (STs) distant from the central genotype within cc269, there was no significant difference for the presence of IS1301 in the IGR of carriage isolates compared to disease isolates. Isolates with insertion of IS1301 in the IGR are not responsible for MenC disease in UK vaccine failures. Novel polymorphisms associated with IS1301 in the IGR of UK MenB isolates do not lead to the resistance phenotype seen for IS1301 in the IGR of MenC isolates.

A Survey of Methods for the Purification of Microbial Flavohemoglobins

Over the past decade, the flavohemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse organisms, including yeasts and fungi but particularly pathogenic bacteria. Flavohemoglobins--the best-characterized class of microbial globin--comprise two domains: a globin domain with a noncovalently bound heme B and a flavin domain with recognizable binding sites for FAD and NAD(P)H. Hmp was first identified in Escherichia coli and now has a clearly defined role in NO biology in that organism: its synthesis is markedly up-regulated by NO, and hmp knockout mutants of E. coli and Salmonella typhimurium are severely compromised for survival in the presence of NO in vitro and in pathogenic lifestyles. In the presence of molecular O2, Hmp catalyzes an oxygenase or denitrosylase reaction in which NO is stoichiometrically converted to nitrate ion, which is relatively innocuous. In this chapter, we present a survey of the methods used to express and purify the flavohemoglobins from diverse microorganisms and describe in more detail three methods developed and used in this laboratory for the E. coli protein. Particular problems are highlighted, particularly (a) the toxic consequences of Hmp overexpression that result from its ability to catalyze partial oxygen reduction and (b) the expression of protein with substoichiometric content of redox-active flavin and heme centers.

Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin–antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.The recalcitrance of many infections to antibiotic treatment may be due to the presence of ‘persisters’, or non-growing, antibiotic-tolerant bacteria. Here, the authors study the structures and functions of aminoacyl-tRNA acetyltransferase toxins, and their roles in persister formation in Salmonella.

Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

Abstract Prokaryotic toxin–antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.