Wastewater sequencing reveals community and variant dynamics of the collective human virome.

Wastewater is a discarded human by-product, but its analysis may help us understand the health of populations. Epidemiologists first analyzed wastewater to track outbreaks of poliovirus decades ago, but so-called wastewater-based epidemiology was reinvigorated to monitor SARS-CoV-2 levels while bypassing the difficulties and pit falls of individual testing. Current approaches overlook the activity of most human viruses and preclude a deeper understanding of human virome community dynamics. Here, we conduct a comprehensive sequencing-based analysis of 363 longitudinal wastewater samples from ten distinct sites in two major cities. Critical to detection is the use of a viral probe capture set targeting thousands of viral species or variants. Over 450 distinct pathogenic viruses from 28 viral families are observed, most of which have never been detected in such samples. Sequencing reads of established pathogens and emerging viruses correlate to clinical data sets of SARS-CoV-2, influenza virus, and monkeypox viruses, outlining the public health utility of this approach. Viral communities are tightly organized by space and time. Finally, the most abundant human viruses yield sequence variant information consistent with regional spread and evolution. We reveal the viral landscape of human wastewater and its potential to improve our understanding of outbreaks, transmission, and its effects on overall population health.

Metagenomic analyses of alcohol induced pathogenic alterations in the intestinal microbiome and the effect of Lactobacillus rhamnosus GG treatment.

Enteric dysbiosis plays an essential role in the pathogenesis of alcoholic liver disease (ALD). Detailed characterization of the alterations in the gut microbiome is needed for understanding their pathogenic role in ALD and developing effective therapeutic approaches using probiotic supplementation. Mice were fed liquid Lieber-DeCarli diet without or with alcohol (5% v/v) for 6 weeks. A subset of mice were administered the probiotic Lactobacillus rhamnosus GG (LGG) from 6 to 8 weeks. Indicators of intestinal permeability, hepatic steatosis, inflammation and injury were evaluated. Metagenomic analysis of the gut microbiome was performed by analyzing the fecal DNA by amplification of the V3-V5 regions of the 16S rRNA gene and large-scale parallel pyrosequencing on the 454 FLX Titanium platform. Chronic ethanol feeding caused a decline in the abundance of both Bacteriodetes and Firmicutes phyla, with a proportional increase in the gram negative Proteobacteria and gram positive Actinobacteria phyla; the bacterial genera that showed the biggest expansion were the gram negative alkaline tolerant Alcaligenes and gram positive Corynebacterium. Commensurate with the qualitative and quantitative alterations in the microbiome, ethanol caused an increase in plasma endotoxin, fecal pH, hepatic inflammation and injury. Notably, the ethanol-induced pathogenic changes in the microbiome and the liver were prevented by LGG supplementation. Overall, significant alterations in the gut microbiome over time occur in response to chronic alcohol exposure and correspond to increases in intestinal barrier dysfunction and development of ALD. Moreover, the altered bacterial communities of the gut may serve as significant therapeutic target for the prevention/treatment of chronic alcohol intake induced intestinal barrier dysfunction and liver disease.

Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM ß-lactamases.

The introduction of extended-spectrum cephalosporins and ß-lactamase inhibitors has driven the evolution of extended-spectrum ß-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter.

The maltose transporter MalFGK(2) of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK(2) and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK(2) during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P(i) release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.

Functional reassembly of the Escherichia coli maltose transporter following purification of a MalF-MalG subassembly.

Taking advantage of a chaperone-like function of MalK, a stable complex of MalF-MalG could be solubilized from the Escherichia coli membrane and purified in high yield in the absence of MalK. This MalF-MalG complex was competent for efficient reassembly of a functional MalFGK(2) maltose transporter complex both in detergent solution and in proteoliposomes.

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex.

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.