- Volume 171, Issue 4, 2025

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has significantly changed the course of the disease in people with cystic fibrosis (CF) (pwCF). The approved triple therapy of elexacaftor, tezacaftor and ivacaftor (ETI), commercially known as Trikafta, increases CFTR channel function, leading to improvements in sweat chloride concentration, exercise capacity, body mass index, lung function and chronic respiratory symptoms. Because of this, the majority of pwCF are living longer and having fewer CF exacerbations. However, colonization with the common CF respiratory pathogens persists and remains a major cause of morbidity and mortality. Here, we review the current literature on the effect of ETI on the respiratory microbiota and discuss the challenges in addressing CF lung infections in the era of these new life-extending therapies.

The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of antimicrobial resistance-related deaths, and novel antimicrobial therapies are urgently required. P. aeruginosa infections are difficult to treat due to the bacterium’s propensity to form biofilms, whereby cells aggregate to form a cooperative, protective structure. Autolysis, the self-killing of bacterial cells, and the bacterial cell-to-cell communication system, quorum sensing (QS), play essential roles in biofilm formation. Strains of P. aeruginosa that have lost the lasI/R QS system commonly develop in patients, and previous studies have characterized distinctive autolysis phenotypes in these strains. Yet, the underlying causes and implications of these autolysis phenotypes remain unknown. This study confirmed these autolysis phenotypes in the PA14 QS mutant strains, ΔlasI and ΔlasR, and investigated the consequences of QS loss and associated autolysis on biofilm formation and antibiotic susceptibility. QS mutants exhibited delayed biofilm formation but ultimately surpassed the wild-type (WT) in biofilm mass. However, the larger biofilm mass of the QS mutants was not reflected in higher live-cell numbers, indicating an altered biofilm structure. Nevertheless, QS mutant biofilms were not more susceptible to antibiotics than the WT. Artificial supplementation of ΔlasI with a QS signal molecule (autoinducer) restored the strain’s QS system without the associated costs of QS, enabling ΔlasI to achieve higher pre-treatment and post-treatment live-cell numbers. Overall, the lack of QS functioning was not detrimental to biofilm antibiotic tolerance, though the artificial disruption of QS may reduce the advantages of QS mutants within in vivo mixed-strain populations. Much remains to be understood regarding the regulation and induction of the autolysis phenotypes observed in these strains, and future research to fully elucidate the control and consequences of autolysis may offer potential for novel antimicrobial therapies.

Despite broad genetic variability, members of the Chlamydiota phylum share a crucial stress response phenotype, the formation of aberrant bodies. However, how this response operates upon exposure to different kinds of stressors is still largely unknown. In Waddlia chondrophila, wcw_0502 RNA levels are upregulated in aberrant bodies induced by iron starvation. Wcw_0502 is a putative type III secretion system (T3SS) effector and has a homologue in every known chlamydial species, regardless of their host. However, the upregulation of the wcw_0502 gene expression upon iron starvation is not conserved in other chlamydial species such as Chlamydia trachomatis, Chlamydia pneumoniae, Simkania negevensis or Estrella lausannensis. Moreover, among all the stressors examined, only heat shock induced a strong upregulation of wcw_0502 and its C. trachomatis homologue, ctl0271. A Controlling Inverted Repeat of Chaperone Expression sequence is present in the promoter region of wcw_0502 and its homologues. We hypothesized that in the absence of stress, the conserved repressor HrcA, in association with the Hsp60 chaperone, binds this sequence and represses transcription. A decreased occupancy of HrcA and Hsp60 at the wcw_0502 promoter region was observed in aberrant bodies induced by iron starvation when compared to reticulate bodies, which may lead to wcw_0502 upregulation. The precise function of this newly described T3SS effector is still unclear. A cystine knot-like domain, a structural feature never described before in bacterial proteins, was found in the C-terminal region of Wcw_0502. This structure is described as highly resistant to proteolytic, chemical and thermic stressors, an advantageous property for a secreted protein with an increased production during stresses that impact protein integrity.

The environmental bacterium Legionella pneumophila, an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here, we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the Acanthamoeba castellanii protozoan infection model. Surprisingly, in trans expression of PsrA partially rescued the growth defect of a dksA1 strain. Whilst in trans expression of DksA expectantly could fully rescue the growth defect of the dksA1 strain, it could also surprisingly rescue the growth defect of a ΔpsrA strain. Conversely, the severe intracellular growth defect of a ΔdksA strain could be rescued by in trans expression of DksA and DksA1, but not PsrA. In vitro phenotypic assays show that either DksA or DksA1 was required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modelling predicts that the DksA1 mutation affects the coordination of Mg2+ into the active site of RNAP, compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, in vitro evidence suggests that the long-chain fatty acid metabolic response is mediated by PsrA together with DksA, inferring a novel regulatory link to the stringent response pathway.

Production of the exopolysaccharide alginate by Azotobacter vinelandii, member of the Pseudomonadaceae family, is positively controlled by the second messenger c-di-GMP. This effect was solely attributed to the role of c-di-GMP in activating the alginate polymerase complex. In this study, the role of c-di-GMP in algD transcription, which encodes the key enzyme for alginate synthesis, was investigated. algD transcription correlated with artificially high or low levels of c-di-GMP. Moreover, FleQ, one of the best-characterized c-di-GMP effectors, was found to exert a negative effect on alginate production and algD transcription, as both increased in a ΔfleQ mutant relative to the wild-type strain or the ΔfleQ/fleQ+ complemented strain. Electrophoretic mobility shift assays (EMSAs) confirmed that FleQ directly binds to the regulatory region of algD, which was consistent with the presence of two FleQ binding sites in the vicinity of the algD RpoS-dependent promoter. In A. vinelandii, c-di-GMP is essential for the expression of alginate C-5 epimerases (AlgE1-6), which are necessary for structuring the envelope of differentiated cells, known as cysts. However, FleQ was not involved in this regulation. Collectively, our results support a model in which algD transcription is under the positive control of c-di-GMP, while FleQ may only partially mediate this effect. In contrast, our study revealed a FleQ-independent regulatory mechanism for the control of A. vinelandii encystment.