Persistence of obligate intracellular pathogens: alternative strategies to overcome host-specific stresses.

In adapting to the intracellular niche, obligate intracellular bacteria usually undergo a reduction of genome size by eliminating genes not needed for intracellular survival. These losses can include, for example, genes involved in nutrient anabolic pathways or in stress response. Living inside a host cell offers a stable environment where intracellular bacteria can limit their exposure to extracellular effectors of the immune system and modulate or outright inhibit intracellular defense mechanisms. However, highlighting an area of vulnerability, these pathogens are dependent on the host cell for nutrients and are very sensitive to conditions that limit nutrient availability. Persistence is a common response shared by evolutionarily divergent bacteria to survive adverse conditions like nutrient deprivation. Development of persistence usually compromises successful antibiotic therapy of bacterial infections and is associated with chronic infections and long-term sequelae for the patients. During persistence, obligate intracellular pathogens are viable but not growing inside their host cell. They can survive for a long period of time such that, when the inducing stress is removed, reactivation of their growth cycles resumes. Given their reduced coding capacity, intracellular bacteria have adapted different response mechanisms. This review gives an overview of the strategies used by the obligate intracellular bacteria, where known, which, unlike model organisms such as E. coli, often lack toxin-antitoxin systems and the stringent response that have been linked to a persister phenotype and amino acid starvation states, respectively.

Tryptophan Availability during Persistence of Chlamydia trachomatis Directly Impacts Expression of Chlamydial Cell Division Proteins.

Chlamydia is an obligate intracellular pathogen with a highly reduced genome devoid of major stress response genes like relA and spoT, which mediate the stringent response. Interestingly, as an intracellular bacterium dependent on its host for nutrients and as a tryptophan (Trp) auxotroph, Chlamydia is very sensitive to Trp starvation, which is induced in vivo by the host cytokine interferon-γ. In response to Trp starvation, Chlamydia enters a viable but nonreplicating state called persistence. A major characteristic of chlamydial persistence is a block in cell division. We hypothesized that cell division is blocked during persistence by the inability to translate Trp-rich cell division proteins. To test this, we first investigated the translation of various cell division proteins under Trp starvation conditions using inducible expression strains. We observed that the Trp-poor protein MurG and the Trp-neutral protein FtsL were still expressed during persistence, while the expression of the Trp-rich proteins Pbp2, RodA, FtsI/Pbp3, and MraY was significantly reduced. As proof of concept for our hypothesis, we compared expression of a wild-type and mutant isoform of RodZ in which its four Trp codons were mutated. These experiments demonstrated that decreased expression of RodZ during persistence was reversed when no Trp was present in the protein, thus directly linking its expression to its Trp content. Together, these experiments indicate that specific cell division proteins are not produced during persistence. For the first time, our data provide a mechanism that explains the inhibition of cell division during chlamydial persistence mediated by Trp starvation.

RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus.

Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin-antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen's overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5'-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3'-end and fine-tunes overall bacterial translation via its 5'-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.

Cross-Regulations between Bacterial Toxin-Antitoxin Systems: Evidence of an Interconnected Regulatory Network?

Toxin-antitoxin (TA) systems are ubiquitous among bacteria and include stable toxins whose toxicity can be counteracted by RNA or protein antitoxins. They are involved in multiple functions that range from stability maintenance for mobile genetic elements to stress adaptation. Bacterial chromosomes frequently have multiple homologues of TA system loci, and it is unclear why there are so many of them. In this review we focus on cross-regulations between TA systems, which occur between both homologous and nonhomologous systems, from similar or distinct types, whether encoded from plasmids or chromosomes. In addition to being able to modulate RNA expression levels, cross-regulations between these systems can also influence their toxicity. This suggests the idea that they are involved in an interconnected regulatory network.

Functionality and cross-regulation of the four SprG/SprF type I toxin-antitoxin systems in Staphylococcus aureus.

Toxin-antitoxin (TA) systems are ubiquitous among bacteria, frequently expressed in multiple copies, and important for functions such as antibiotic resistance and persistence. Type I TA systems are composed of a stable toxic peptide whose expression is repressed by an unstable RNA antitoxin. Here, we investigated the functionalities, regulation, and possible cross-talk between three core genome copies of the pathogenicity island-encoded 'sprG1/sprF1' type I TA system in the human pathogen Staphylococcus aureus. Except for SprG4, all RNA from these pairs, sprG2/sprF2, sprG3/sprF3, sprG4/sprF4, are expressed in the HG003 strain. SprG2 and SprG3 RNAs encode toxic peptides whose overexpression triggers bacteriostasis, which is counteracted at the RNA level by the overexpression of SprF2 and SprF3 antitoxins. Complex formation between each toxin and its cognate antitoxin involves their overlapping 3' ends, and each SprF antitoxin specifically neutralizes the toxicity of its cognate SprG toxin without cross-talk. However, overexpression studies suggest cross-regulations occur at the RNA level between the SprG/SprF TA systems during growth. When subjected to H2O2-induced oxidative stress, almost all antitoxin levels dropped, while only SprG1 and SprF1 were reduced during phagocytosis-induced oxidative stress. SprG1, SprF1, SprF2, SprG3 and SprF3 levels also decrease during hyperosmotic stress. This suggests that novel SprG/SprF TA systems are involved in S. aureus persistence.