Biology subjects

Henrichfreise, B.

Publications and source records attributed to Henrichfreise, B..

4 recordsLinked to original sources

bioRxiv2025

Characterization of the Cell Division-Associated Peptidoglycan Amidase AmiA of Chlamydia trachomatis

Chlamydia is an obligate intracellular bacterium that differentiates between infectious, non-dividing EBs and non-infectious, dividing RBs. Pathogenic Chlamydia species are unusual in lacking a peptidoglycan sacculus, yet they do synthesize a transient and localized peptidoglycan structure at the divisome of the RB during their polarized division process. Although several studies have described the components of the chlamydial divisome necessary to generate peptidoglycan at a specific site on the membrane, less is understood about how the peptidoglycan structure is degraded to allow for the daughter cell to form and the division process to complete. Amidases are key components of the cell wall in model system bacteria as they catalyze the degradation and remodeling of peptidoglycan, including in the division septum. Here, we characterized the cell division-associated amidase, AmiA_Ct, of Chlamydia trachomatis both in vitro and in vivo. Our in vitro data show that AmiA_Ct is a bona fide, metal-dependent amidase capable of cleaving peptidoglycan. AmiA_Ct complemented an E. coli amidase deficient strain and supported the growth and separation of daughter cells. To assess the function of AmiA_Ct in C. trachomatis, we generated a transformant strain carrying an inducible CRISPR interference system targeting the amiA gene. Knocking down expression of amiA resulted in altered bacterial morphology, a reduction in infectious EBs, and the accumulation of peptidoglycan in the organisms. These data indicate a critical function for AmiA_Ct in the unique cell division process of Chlamydia. ImportancePeptidoglycan is an important structural cell wall polymer that serves to give bacteria their shape and resistance to changes in extracellular solute concentrations. For Chlamydia trachomatis, an obligate intracellular pathogen that divides within a host cell, peptidoglycan is only used for cell division and is not a component of its cell wall. In this study, we characterize the function of a chlamydial amidase that helps degrade peptidoglycan during cell division. We show a critical function for amidase activity in facilitating changes to the peptidoglycan structure during chlamydial cell division that support normal growth and development of this pathogenic bacterium.

microbiology↗

bioRxiv2025

Chlamydia trachomatis restricts signaling through NOD2 until late in the pathogen's developmental cycle

Pathogenic chlamydial species restrict their peptidoglycan (PG) to the division septum of their replicative forms. PG is a microbe-associated molecular pattern (MAMP) and two of its major pattern recognition receptors in human cells are nucleotide-binding oligomerization domain-containing proteins 1 and 2 (NOD1 and NOD2, respectively). It has been proposed that this unique morphological feature is evidence of pathoadaptation by the microbe, permitting PG-dependent cell division while also reducing the bacteriums recognition by innate immune receptors. Chlamydia trachomatis-infected cells activate NOD1 signaling within 8-12 hours of exposure to the bacterium, roughly coinciding with the microbes transition from its infectious to replicative forms. Here we report that, unlike NOD1 signaling, Chlamydia-induced NOD2 signaling does not occur until later in the pathogens developmental cycle. Both C. trachomatis and the related murine pathogen Chlamydia muridarum signal late in infection in HEK293 reporter cell lines expressing either human or murine-derived NOD2 receptors. NOD2 signaling can be modulated by disruption of the chlamydial amidase enzyme, AmiACT, interrupting the microbes developmental cycle, and treatment with inhibitors of lipooligosaccharide or peptidoglycan biosynthesis / assembly. These results mirror prior observations with Chlamydia-induced TLR9 signaling, leading us to hypothesize that Chlamydia-induced NOD2 signaling results from lytic events that occur sporadically during the transition between the pathogens developmental forms. Given our finding that pre-treating cells with NOD2-stimulatory ligands reduces chlamydial inclusion size and delays the developmental cycle, we hypothesize that the microbe preferentially degrades its PG during development to reduce the generation of NOD2 ligands.

microbiology↗

bioRxiv2022

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs. Author SummaryFree-living bacteria are wrapped by a peptidoglycan cell wall. This envelope provides shape and protects from osmotic stress, while also triggering human immune defense. Chlamydial pathogens live inside human cells and dispensed with a cell wall in their isotonic niche. Instead, they produce a transient peptidoglycan ring that is essentially involved in cell division. Recycling of peptidoglycan is likely important for chlamydiae to maintain a complete cycle of peptidoglycan ring synthesis and cell division. Moreover, the process may help to save energy and to protect from recognition through the immune system. Despite a potentially central role in cell viability and pathogenicity almost nothing is known about recycling of peptidoglycan in chlamydiae. The minimalist organisms lack all enzymes known to catalyze the process in model organism Escherichia coli. Here, we found an NlpC/P60 enzyme to serve a critical step in decomposing peptidoglycan ring derived peptides in Chlamydia trachomatis. Our data indicate that the peptidase recycles energy cost intensive components and breaks down the minimal recognition motif of innate immune factor NOD1. We also identified a natural lead compound to inhibit the NlpC/P60 enzyme opening the way for innovative anti-chlamydial drug development at the intersection of energy recovery, cell division, and immune evasion.

microbiology↗

bioRxiv2022

In vivo and In vitro Characterization of the ClpC AAA+ ATPase of Chlamydia trachomatis

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and in vivo approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated {beta}-casein. In vivo experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, the in vivo data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of anti-chlamydial agents. SignificanceChlamydia trachomatis is an obligate intracellular pathogen and the worlds leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new anti-chlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for anti-chlamydial compounds.

microbiology↗