Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using , we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31. The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as , but other proteins have critical regulatory functions that may be unique to the actinomycetes.

Salmonella enterica serovar Typhimurium is a gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SSs) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of Salmonella by cytosolic immune sensors. Upon detecting translocated Salmonella ligands, these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to proinflammatory cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella . While these processes are studied quite well in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human intestinal epithelial cell lines and organoids. Additionally, the NLRP3 inflammasome and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a partial requirement for caspase-1, and a necessity for caspase-4 and GSDMD pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4, and also do not use NLRP3/ASC. Instead, they primarily use caspases-1 and -4 to mediate early inflammasome responses to SPI-1-expressing Salmonella .