Mechanisms of human adenovirus inactivation by sunlight and UVC light as examined by quantitative PCR and quantitative proteomics.

Human adenoviruses (HAdV) are important pathogens in both industrialized and developing nations. HAdV has been shown to be relatively resistant to monochromatic UVC light. Polychromatic UVC light, in contrast, is a more effective means of disinfection, presumably due to the involvement of viral proteins in the inactivation mechanism. Solar disinfection of HAdV, finally, is only poorly understood. In this paper, the kinetics and mechanism of HAdV inactivation by UVC light and direct and indirect solar disinfection are elucidated. PCR and mass spectrometry were employed to quantify the extent of genome and protein degradation and to localize the affected regions in the HAdV proteins. For this purpose, we used for the first time an approach involving stable isotope labeling by amino acids in cell culture (SILAC) of a human virus. Inactivation by UVC light and the full sunlight spectrum were found to efficiently inactivate HAdV, whereas UVA-visible light only caused inactivation in the presence of external sensitizers (indirect solar disinfection). Genome damage was significant for UVC but was less important for solar disinfection. In contrast, indirect solar disinfection exhibited extensive protein degradation. In particular, the fiber protein and the amino acids responsible for host binding within the fiber protein were shown to degrade. In addition, the central domain of the penton protein was damaged, which may inhibit interactions with the fiber protein and lead to a disruption of the initial stages of infection. Damage to the hexon protein, however, appeared to affect only regions not directly involved in the infectious cycle.

Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity.

Oxidative processes are often harnessed as tools for pathogen disinfection. Although the pathways responsible for bacterial inactivation with various biocides are fairly well understood, virus inactivation mechanisms are often contradictory or equivocal. In this study, we provide a quantitative analysis of the total damage incurred by a model virus (bacteriophage MS2) upon inactivation induced by five common virucidal agents (heat, UV, hypochlorous acid, singlet oxygen, and chlorine dioxide). Each treatment targets one or more virus functions to achieve inactivation: UV, singlet oxygen, and hypochlorous acid treatments generally render the genome nonreplicable, whereas chlorine dioxide and heat inhibit host-cell recognition/binding. Using a combination of quantitative analytical tools, we identified unique patterns of molecular level modifications in the virus proteins or genome that lead to the inhibition of these functions and eventually inactivation. UV and chlorine treatments, for example, cause site-specific capsid protein backbone cleavage that inhibits viral genome injection into the host cell. Combined, these results will aid in developing better methods for combating waterborne and foodborne viral pathogens and further our understanding of the adaptive changes viruses undergo in response to natural and anthropogenic stressors.

Protein oxidation and aggregation in UVA-irradiated Escherichia coli cells as signs of accelerated cellular senescence.

Solar disinfection (SODIS) is a simple drinking water treatment method that improves microbiological water quality where other means are unavailable. It makes use of the deleterious effect of solar irradiation on pathogenic microbes and viruses. A positive impact on health has been documented in several epidemiological studies. However, the molecular mechanisms damaging cells during this simple treatment are not yet fully understood. Here we show that protein damage is crucial in the process of inactivation by sunlight. Protein damages in UVA-irradiated Escherichia coli cells have been evaluated by an immunoblot method for carbonylated proteins and an aggregation assay based on semi-quantitative proteomics. A wide spectrum of structural and enzymatic proteins within the cell is affected by carbonylation and aggregation. Vital cellular functions like the transcription and translation apparatus, transport systems, amino acid synthesis and degradation, respiration, ATP synthesis, glycolysis, the TCA cycle, chaperone functions and catalase are targeted by UVA irradiation. The protein damage pattern caused by SODIS strongly resembles the pattern caused by reactive oxygen stress. Hence, sunlight probably accelerates cellular senescence and leads to the inactivation and finally death of UVA-irradiated cells.

The respiratory chain is the cell's Achilles' heel during UVA inactivation in Escherichia coli.

Solar disinfection (SODIS) is used as an effective and inexpensive tool to improve the microbiological quality of drinking water in developing countries where no other means are available. Solar UVA light is the agent that inactivates bacteria during the treatment. Damage to bacterial membranes plays a crucial role in the inactivation process. This study showed that even slightly irradiated cells (after less than 1 h of simulated sunlight) were strongly affected in their ability to maintain essential parts of their energy metabolism, in particular of the respiratory chain (activities of NADH oxidase, succinate oxidase and lactate oxidase were measured). The cells' potential to generate ATP was also strongly inhibited. Many essential enzymes of carbon metabolism (glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase) and defence against oxidative stress (catalases and glutathione-disulfide reductase) were reduced in their activity during SODIS. The work suggests that damage to membrane enzymes is a likely cause of membrane dysfunction (loss of membrane potential and increased membrane permeability) during UVA irradiation. In this study, the first targets on the way to cell death were found to be the respiratory chain and F(1)F(0) ATPase.

Solar disinfection (SODIS) and subsequent dark storage of Salmonella typhimurium and Shigella flexneri monitored by flow cytometry.

Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation: apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m(-2)) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (<400 W m(-2)).

Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.