The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of post lysis degradation. Here we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858), and a third strain containing a mutation in Type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF− C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity, and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein Lamin-associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.

bThe second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems. Since its discovery in 1987 as an allosteric activator of bacterial cellulose synthesis (1), cyclic diguanylate (c-di-GMP) has been shown to be a remarkably important signaling molecule across diverse bacterial species, controlling a multitude of behaviors and processes, including biofilm formation, motility, virulence, cell cycle progression, and differentiation (2-4). An important feature of c-di-GMP regulation is the ability of this signal to control critical lifestyle transitions, such as motile-sessile transitions (e.g., planktonic to biofilm), which are undertaken by many bacterial species (3, 5, 6). Generally speaking, elevated levels of c-di-GMP promote sessile lifestyles such as biofilm formation; in contrast, low levels of c-di-GMP are associated with motility (3, 6). Intracellular levels of this dinucleotide are controlled by opposing activities of enzymes that synthesize c-di-GMP (GGDEF domaincontaining diguanylate cyclases [DGCs]) and those that cleave this signaling molecule (EAL-or HD-GYP domain-containing phosphodiesterases [PDEs]) (3,4,(7)(8)(9)(10)(11).More recently, studies focused on how cells respond to changing c-di-GMP levels, indicated that this signaling network relies upon proteins or RNA molecules, known as c-di-GMP effectors (or receptors), which bind c-di-GMP and whose output functions are altered due to c-di-GMP-mediated structural changes (3,4,12). A number of distinct effectors have been identified and classified based on their c-di-GMP-binding motif. The PilZ class of c-di-GMP effector proteins is one of the best-studied class...

We compared the performance of the Abbott BinaxNOW COVID-19 Antigen Card to a standard RT-PCR assay (ThermoFisher TaqPath COVID-19 Combo Kit) for the detection of SARS-CoV-2 in 2,645 asymptomatic students presenting for screening at the University of Utah. SARS-CoV-2 RNA was detected in 1.7% of the study participants by RT-PCR. BinaxNOW identified 24 infections but missed 21 infections that were detected by RT-PCR. The analytical sensitivity (positive agreement) and analytical specificity (negative agreement) for the BinaxNOW was 53.3% and 100%, respectively when compared against the RT-PCR assay. The median cycle threshold (Ct) value in the specimens that had concordant positive BinaxNOW antigen result was significantly lower compared to those that were discordant (Ct 17.6 vs. 29.6; p < 0.001). In individuals with presumably high viral loads (Ct < 23.0), a 95.8% positive agreement was observed between the RT-PCR assay and BinaxNOW. Due to the possibility of false negative results, caution must be taken when utilizing rapid antigen testing for screening asymptomatic individuals.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.