Assessment of the potential occurrence of Cryptosporidium species in various water sources in Sharqia Governorate, Egypt.

Cryptosporidium species are enteric apicomplexan parasites associated with diarrhoeal disease in humans and animals globally. Waterborne outbreaks resulting from contamination with the infective oocysts are common worldwide. Updated reports on waterborne protozoal infections are needed to identify emerging pathogens and susceptible populations. Therefore, this study aimed to assess the current profile of Cryptosporidium contamination of various water sources in Sharqia Governorate, Northeastern Egypt. For this purpose, eighty samples were collected from five different water types (canal, tap, tank, filtered, and groundwater), distributed in four major cities (El-Hessenia, Fakous, Zagazig, and Belbies) in Sharqia Governorate. All water samples were examined using conventional microscopy, ELISA, and real-time PCR (RT-PCR) techniques. Based on microscopic analysis, the Cryptosporidium protozoan was identified in 25% of the tested water samples. The RT-PCR assay has allowed for the quantification of Cryptosporidium oocysts in different types of water. Canal water exhibited the highest Cryptosporidium contamination levels (mean = 85.15 oocysts/L), followed by water tanks (mean = 12.031 oocysts/L). The study also provided a comparative evaluation of ELISA and RT-PCR for the diagnosis of Cryptosporidium infection. RT-PCR performed better than ELISA in terms of analytical accuracy (97.50% vs. 86.25%) and specificity (100% vs. 83.33%). However, ELISA showed a higher sensitivity (95.00% vs. 90.00%) for Cryptosporidium recovery. Our findings could serve as a platform for further investigations into the potential risks associated with water contamination in Sharqia Governorate. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01675-1.

ATP hydrolysis tunes specificity of a AAA+ protease.

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. In vitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.

Degradation of Lon in Caulobacter crescentus.

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae.

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Regulated Proteolysis in Bacteria.

Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.