Dodd, C. E. | bioRxiv Science

bioRxiv2022

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and - 9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection. Author SummaryIntracellular bacterial pathogens cause many of the worlds most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a "pattern of pathogenesis" that triggers antimicrobial immune responses. Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogens Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Mycobacterium tuberculosis (Mtb), and were only modestly susceptible to chronic Mtb infection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.

immunology↗

bioRxiv2020

Ceragenins and antimicrobial peptides kill bacteria through distinct mechanisms

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responds similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicates that while some bacterial responses to ceragenins overlap with those induced by naturally-occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCEThe development of novel antibiotics is essential since the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. Development of naturally-occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation and have been associated with low levels of resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

microbiology↗