Identification and genome sequencing of an influenza H3N2 variant in wastewater from elementary schools during a surge of influenza A cases in Las Vegas, Nevada.

Real-time surveillance of infectious diseases at schools or in communities is often hampered by delays in reporting due to resource limitations and infrastructure issues. By incorporating quantitative PCR and genome sequencing, wastewater surveillance has been an effective complement to public health surveillance at the community and building-scale for pathogens such as poliovirus, SARS-CoV-2, and even the monkeypox virus. In this study, we asked whether wastewater surveillance programs at elementary schools could be leveraged to detect RNA from influenza viruses shed in wastewater. We monitored for influenza A and B viral RNA in wastewater from six elementary schools from January to May 2022. Quantitative PCR led to the identification of influenza A viral RNA at three schools, which coincided with the lifting of COVID-19 restrictions and a surge in influenza A infections in Las Vegas, Nevada, USA. We performed genome sequencing of wastewater RNA, leading to the identification of a 2021-2022 vaccine-resistant influenza A (H3N2) 3C.2a1b.2a.2 subclade. We next tested wastewater samples from a treatment plant that serviced the elementary schools, but we were unable to detect the presence of influenza A/B RNA. Together, our results demonstrate the utility of near-source wastewater surveillance for the detection of local influenza transmission in schools, which has the potential to be investigated further with paired school-level influenza incidence data.

Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon.

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

Soluble extracellular Klotho decreases sensitivity to cigarette smoke induced cell death in human lung epithelial cells.

Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the US and is associated with an abnormal inflammatory response to cigarette smoke (CS). Exposure to CS induces oxidative stress and can result in cellular senescence in the lung. Cellular senescence can then lead to decreased proliferation of epithelial cells, the destruction of alveolar structure and pulmonary emphysema. The anti-aging gene, klotho, encodes a membrane bound protein that has been shown to be a key regulator of oxidative stress and cellular senescence. In this study the role of Klotho (KL) with regard to oxidative stress and cellular senescence was investigated in human pulmonary epithelial cells exposed to cigarette smoke. Individual clones that stably overexpress Klotho were generated through retroviral transfection and geneticin selection. Klotho overexpression was confirmed through RT-qPCR, Western blotting and ELISA. Compared to control cells, constitutive Klotho overexpression resulted in decreased sensitivity to cigarette smoke induced cell death in vitro via a reduction of reactive oxygen species and a decrease in the expression of p21. Our results suggest that increasing Klotho level in pulmonary epithelial cells may be a promising strategy to reduce cellular senescence and mitigate the risk for the development of COPD.