Macrophage cell lines and murine infection by Salmonella Typhi L-form bacteria

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic-resistance genes is well-established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as the one due to treatment with bacterial cell-wall inhibiting antibiotics like ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin. This is different from in vitro drug testing where the organism is exposed to fixed drug concentrations from the beginning till the end. To mimic the mode of antibiotic exposure of microorganisms in the tissues, we cultured the wild type, ampicillin-sensitive Salmonella Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that exhibited several features of the so-called L-form of bacteria, such as the absence of cell wall, altered shape and slower growth rate compared with the parental strain. Studies on the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental strain. These results suggested that L-form generation following initiation of antibiotic treatment could lead to drug escape of S. Typhi and direct spread to new cells (macrophages), which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through faeco-oral route, highlighting the need for new approaches to decrease the reservoir of infection.

Glutathione Reductase Encoding Gene (gor) is Associated with Oxidative Stress and Antibiotic Susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major causative agent of the hospital- and community-acquired infections. These infections are often antibiotic resistant and difficult to treat. Several intrinsic and acquired resistance mechanisms to antibiotics have reported in P. aeruginosa. Recently, oxidative- stress-scavenging-systems have suggested as a possible intrinsic resistance mechanism to antibiotics because oxidative stresses induced by bactericidal antibiotics contribute to bacterial killing effects. However, this remains controversial such that further clarification is required. Glutathione reductase is a key enzyme in the maintenance of the optimum level of intracellular glutathione-redox potential to ensure normal functioning of cellular processes including the detoxification of oxidative stress. In this study, the role of a glutathione-reductase-encoding gene (gor) in oxidative stress and antibiotic susceptibility was determined in P. aeruginosa. Results showed that a gor-mutant strain was more susceptible to hydrogen peroxide (but not superoxide) than the parental strain and 100% of cells were killed with 0.01% hydrogen peroxide while the parental strain survived at the same concentration of hydrogen peroxide. The gor-mutant strain was also more susceptible to carbenicillin, chloramphenicol, ciprofloxacin, and tetracycline than the parental strain, which was confirmed by bacterial killing-kinetics. These results suggest that the gor gene is associated with oxidative stress and susceptibility to bactericidal as well as bacteriostatic antibiotics and that the oxidative-stress-scavenging-systems may be a possible drug-target for multidrug resistant P. aeruginosa.

Rhoptry kinase protein 39 (ROP39) is a novel factor that recruits host mitochondria to the parasitophorous vacuole of Toxoplasma gondii

ABSTRACT Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain.

Metabolic Plasticity Aids Amphotropism of Coxiella burnetii

Coxiella burnetii , the causative agent of Query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms (e.g., mammals and arthropods) and cell types. This amphotropic nature of C. burnetii , in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, Cb Δ pckA, unable to undergo gluconeogenesis. Both the parental strain and Cb Δ pckA exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, Interstitial Fluid-modeled Medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by Cb Δ pckA in specific cell lines. Cb Δ pckA was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen.

Host lung environment limits Aspergillus fumigatus germination through a SskA-dependent signaling response

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential; but the mechanism(s) regulating A. fumigatus germination in the airways are unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine lung based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named Lung Homogenate Evolved (LH-EVOL)). We identified a potential loss of function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decrease ability to induce the SakA-dependent stress pathway. In support of the whole genome variant analyses, sskA, sakA, or mpkC loss of function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA MAPK pathway and drives enhanced disease initiation and inflammation in the lungs.

c-di-AMP signaling is required for bile salts resistance and long-term colonization by Clostridioides difficile

ABSTRACTTo cause disease, the important human enteropathogen Clostridioides difficile must colonize the gastro-intestinal tract but little is known on how this organism senses and responds to the harsh host environment to adapt and multiply. Nucleotide second messengers are signaling molecules used by bacteria to respond to changing environmental conditions. In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. In a colonization mouse model, a strain lacking GdpP, a c-di-AMP degrading enzyme, failed to persist in the gut in contrast to the parental strain. We identified OpuR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter OpuC. Interestingly, an opuR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while an opuCA mutant is more susceptible A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host.One Sentence Summaryc-di-AMP is an essential regulatory molecule conferring resistance to osmotic and bile salt stresses by controlling osmolyte uptake and contributing to gut persistence in the human enteropathogen Clostridioides difficile.

Longitudinal Relations Between Parental Strain, Parent–Child Relationship Quality, and Child Well-Being During the Unfolding COVID-19 Pandemic

As COVID-19 sweeps across the globe, scientists have identified children and families as possibly particularily vulnerable populations. The present study employed a developmental framework with two measurement points (the first at the peak of the lockdown restrictions (N = 2,921), the second after restrictions had been majorly loosened (N = 890)) to provide unique insights into the relations between parental strain, child well-being, and child problem behavior. Cross-lagged panel analyses revealed longitudinal effects of child well-being and problem behavior at T1 on parental strain at T2 with parent–child relationship quality as a moderator. True intraindividual change models showed that decreases in parental strain between measurement points predicted increases in child well-being and decreases in child problem behavior. Thus, the present research points to parental stress coping and child emotional adjustment as promising avenues for professionals and policy makers in their efforts to ensure child and family well-being throughout the pandemic.

Improvement of FK506 production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis

Background FK506, a macrolide mainly with immunosuppressive activity, can be produced by various Streptomyces strains. However, one of the major challenges in the fermentation of FK506 is its insufficient production, resulting in high fermentation costs and environmental burdens. Herein, we tried to improve its production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis. Results First, basing on the genome sequencing and analysis, putative competitive pathways were deleted. A better parental strain L19-2 with increased FK506 production from 140.3 to 170.3 mg/L and a cleaner metabolic background was constructed. Subsequently, the FK506 biosynthetic gene cluster was refactored by in-situ promoter-substitution strategy basing on the regulatory circuits. This strategy enhanced transcription levels of the entire FK506 biosynthetic gene cluster in a fine-tuning manner and dramatically increased the FK506 production to 410.3 mg/mL, 1.41-fold higher than the parental strain L19-2 (170.3 mg/L). Finally, the FK506 production was further increased from 410.3 to 603 mg/L in shake-flask culture by adding L-isoleucine at a final concentration of 6 g/L. Moreover, the potential of FK506 production capacity was also evaluated in a 15-L fermenter, resulting in the FK506 production of 830.3 mg/L. Conclusion From the aspects of competitive pathways, refactoring of the FK506 biosynthetic gene cluster and nutrients-addition, a strategy for hyper-production and potentially industrial application of FK506 was developed and a hyper-production strain L19-9 was constructed. The strategy presented here can be generally applicable to other Streptomyces for improvement of FK506 production and streamline hyper-production of other valuable secondary metabolites.

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood.Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity.Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection.Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric.

The mia mutants of Streptomyces albus J1074 are prone to translational errors and susceptible to certain stressors

Streptomyces albus J1074 has been established by us as a convenient model to study different aspects of tRNALeuUAA-dependent regulatory mechanisms, that take place in genus Streptomyces. These mechanisms are important for proper morphological and physiological transitions of streptomycete colonies, such as the onset of antibiotic production in stationary phase of growth. The genes for post-transcriptional modification of adenosine residue in 37th position of tRNAXXA family (so called mia genes) were shown to be important for the aforementioned processes, most likely because they impact tRNALeuUAA among other tRNAs. Our results were largely consistent with what is known about mia mutations in the other model systems, such as yeast and enterobacteria. Nevertheless, we also revealed several differences from the model systems, such as decreased susceptibility to hydrogen peroxide. This prompted us to look deeper into the behavior of the mia mutants, particularly their response to different stress factors. Here we report that S. albus mia mutants exhibit increased mistranslation rate as compared to their parental strain. These mutants are more susceptible than the parental strain to disulfide stress inducer diamide and DNA repair stressor caffeine. In summary, although the deficiency in certain tRNA modification appears to cause identical or very similar response (such as elevated mistranslation) across all so far studied bacterial systems, it also induces species- or genus-specific effects (such as disparate effects on H2O2 susceptibility). These differences could be attributed to the peculiarities of organization/function of regulatory pathway governing the response to a given stress. The observed results are further discussed in the wider context of the role of tRNA modification pathway in bacterial biology.