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IntroductionSARS-CoV-2 pandemic still poses a significant burden on global health and economy, especially for symptoms persisting beyond the acute disease. COVID-19 manifests with various degrees of severity and the identification of early biomarkers capable of stratifying patient based on risk of progression could allow tailored treatments.MethodsWe longitudinally analyzed 67 patients, classified according to a WHO ordinal scale as having Mild, Moderate, or Severe COVID-19. Peripheral blood samples were prospectively collected at hospital admission and during a 6-month follow-up after discharge. Several subsets and markers of the innate and adaptive immunity were monitored as putative factors associated with COVID-19 symptoms.ResultsMore than 50 immunological parameters were associated with disease severity. A decision tree including the main clinical, laboratory, and biological variables at admission identified low NK-cell precursors and CD14+CD91+ monocytes, and high CD8+ Effector Memory T cell frequencies as the most robust immunological correlates of COVID-19 severity and reduced survival. Moreover, low regulatory B-cell frequency at one month was associated with the susceptibility to develop long COVID at six months, likely due to their immunomodulatory ability.DiscussionThese results highlight the profound perturbation of the immune response during COVID-19. The evaluation of specific innate and adaptive immune-cell subsets allows to distinguish between different acute and persistent COVID-19 symptoms.

ABSTRACT Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression. IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. IMPORTANCE Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P. aeruginosa infection. These results now provide a basis for mapping genomic regions underlying host susceptibility to P. aeruginosa infection.

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1beta cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.

Mycobacterium abscessus causes severe disease in patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene regulation. We show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough strain, increased pro-inflammatory signaling and virulence in multiple infection models, and increased resistance to antibiotics. Examination of clinical isolate cohorts identified isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of mutations found in clinical isolates. Over 200 genes were differentially expressed in the deletion mutant. Strains with the clinical B11 mutations showed expression trends similar to the deletion mutant, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with B11 functioning as a negative regulator that represses translation via base-pairing to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences. Intriguingly, genes upregulated in the absence of B11 included components of the ESX-4 secretion system, critical for M. abscessus virulence. Many of these genes had B11-complementary sequences at their RBSs, which we show is sufficient to mediate repression by B11 through direct binding. Altogether, our data show that B11 acts as a direct negative regulator and mediates (likely indirect) positive regulation with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains is consistent with the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. This is the first report on an sRNA role in M. abscessus.

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, expressed on the apical surface of epithelial cells. CFTR absence/dysfunction results in ion imbalance and airway surface dehydration that severely compromise the CF airway microenvironment, increasing infection susceptibility. Recently, novel therapies aimed at correcting the basic CFTR defect have become available, leading to substantial clinical improvement of CF patients. The restoration or increase of CFTR function affects the airway microenvironment, improving local defence mechanisms. CFTR modulator drugs might therefore affect the development of chronic airway infections and/or improve the status of existing infections in CF. Thus far, however, the full extent of these effects of CFTR-modulators, especially in the long-term remains still unknown. This review aims to provide an overview of current evidence on the potential impact of CFTR modulators on airway infections in CF. Their role in affecting CF microbiology, the susceptibility to infections as well as the potential efficacy of their use in preventing/decreasing the development of chronic lung infections and the recurrent acute exacerbations in CF will be critically analysed.