The presence of cystic fibrosis-related diabetes modifies the sputum microbiome in cystic fibrosis disease.

Cystic fibrosis-related diabetes (CFRD) affects 40%-50% of adults with CF and is associated with a decline in respiratory health. The microbial flora of the lung is known to change with the development of CF disease, but how CFRD affects the microbiome has not been described. We analyzed the microbiome in sputa from 14 people with CF, 14 with CFRD, and two who were classed as pre-CFRD by extracting DNA and amplifying the variable V3-V4 region of the microbial 16S ribosomal RNA gene by PCR. Sequences were analyzed and sources were identified to genus level. We found that the α-diversity of the microbiome using Shannon's diversity index was increased in CFRD compared with CF. Bray Curtis dissimilarity analysis showed that there was separation of the microbiomes in CF and CFRD sputa. The most abundant phyla identified in the sputum samples were Firmicutes and Proteobacteria, Actinobacteriota and Bacteroidota, and the ratio of Firmicutes/Bacteroidota was reduced in CFRD compared with CF. Pseudomonas, Azhorizophilus, Porphyromonas, and Actinobacillus were more abundant in CFRD compared with CF, whereas Staphylococcus was less abundant. The relative abundance of these genera did not correlate with age; some correlated with a decline in FEV1/FVC but all correlated with hemoglobin A1C (HbA1c) indicating that development of CFRD mediates further changes to the respiratory microbiome in CF.NEW & NOTEWORTHY Cystic fibrosis-related diabetes (CFRD) is associated with a decline in respiratory health. We show for the first time that there was a change in the sputum microbiome of people with CFRD compared with CF that correlated with markers of raised blood glucose.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Emergence of phenotypic and genotypic antimicrobial resistance in Mycobacterium tuberculosis.

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of resistance did not occur at concentration-time profiles mimicking the granuloma. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides an improved rational for drug and dose selection although further work to understand the underlying mechanisms is needed to improve the drug development pipeline.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.