Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB).

The similarities between fungal and mammalian cells pose inherent challenges for the development of treatments for fungal infections, due to drug crossover recognition of host drug targets by antifungal agents. Thus, there are a limited number of drug classes available for treatment. Treatment is further limited by the acquisition and dissemination of antifungal resistance which contributes to the urgent need of new therapies. Polyhexamethylene biguanide (PHMB) is a cationic antimicrobial polymer with bactericidal, parasiticidal and fungicidal activities. The antifungal mechanism of action appears to involve preferential mechanical disruption of microbial cell structures, offering an alternative to conventional antifungals. However, the antifungal mechanisms have been little studied. The aim of this study was to characterise PHMB's activities on selected yeast (Saccharomyces cerevisiae, Candida albicans) and filamentous fungal species (Fusarium oxysporum, Penicillium glabrum). Fungal membrane disruption, cell entry and intracellular localisation activities of PHMB were evaluated using viability probe entry and polymer localisation studies. We observed that PHMB initially permeabilises fungal cell membranes and then accumulates within the cytosol. Once in the cytosol, it disrupts the nuclear membrane, leading to DNA binding and fragmentation. The electrostatic interaction of PHMB with membranes suggests other intracellular organelles could be potential targets of its action. Overall, the results indicate multiple antifungal mechanisms, which may help to explain its broad-spectrum efficacy. A better understanding of PHMB's mechanism(s) of action may aid the development of improved antifungal treatment strategies.

Defining the Genes Required for Survival of Mycobacterium bovis in the Bovine Host Offers Novel Insights into the Genetic Basis of Survival of Pathogenic Mycobacteria.

Tuberculosis has severe impacts on both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human-adapted species, and Mycobacterium bovis, the animal-adapted species, is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models; however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study, we utilize transposon insertion sequencing in M. bovis AF2122/97 to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, phthiocerol dimycocerosate (PDIM) synthesis, mycobactin synthesis, and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis H37Rv, phoPR is required by M. bovis AF2122/97 in vivo despite the known defect in signaling through this system. Comparison to studies performed in species that are able to use carbohydrates as an energy source, such as M. bovis BCG and M. tuberculosis, suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon) and oxidation (hsd). We report a good correlation with existing mycobacterial virulence functions but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism, and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. IMPORTANCE This is the first report of the genetic requirements of an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC) in a natural host. M. bovis has devastating impacts on cattle, and bovine tuberculosis is a considerable economic, animal welfare, and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identify several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.