Gain-of-function variants of FtsA form diverse oligomeric structures on lipids and enhance FtsZ protofilament bundling.

Escherichia coli requires FtsZ, FtsA and ZipA proteins for early stages of cell division, the latter two tethering FtsZ polymers to the cytoplasmic membrane. Hypermorphic mutants of FtsA such as FtsA* (R286W) map to the FtsA self-interaction interface and can bypass the need for ZipA. Purified FtsA forms closed minirings on lipid monolayers that antagonize bundling of FtsZ protofilaments, whereas FtsA* forms smaller oligomeric arcs that enable bundling. Here, we examined three additional FtsA*-like mutant proteins for their ability to form oligomers on lipid monolayers and bundle FtsZ. Surprisingly, all three formed distinct structures ranging from mostly arcs (T249M), a mixture of minirings, arcs and straight filaments (Y139D) or short straight double filaments (G50E). All three could form filament sheets at higher concentrations with added ATP. Despite forming these diverse structures, all three mutant proteins acted like FtsA* to enable FtsZ protofilament bundling on lipid monolayers. Synthesis of the FtsA*-like proteins in vivo suppressed the toxic effects of a bundling-defective FtsZ, exacerbated effects of a hyper-bundled FtsZ, and rescued some thermosensitive cell division alleles. Together, the data suggest that conversion of FtsA minirings into any type of non-miniring oligomer can promote progression of cytokinesis through FtsZ bundling and other mechanisms.

Development of Mycoplasma pneumoniae biofilms in vitro and the limited role of motility.

Mycoplasma pneumoniae is a bacterial pathogen of humans that is a major causative agent of chronic respiratory disease. M. pneumoniae infections often recur even after successful treatment of symptoms with antibiotics, and resistance to antibiotics is increasing worldwide, with nearly complete resistance in some places. Although biofilms often contribute to chronicity and resistance, M. pneumoniae biofilms remain poorly characterized. Scanning electron microscopy revealed that cells of wild-type (WT) M. pneumoniae strain M129 biofilms, as well as mutants II-3 and II-3R, in vitro became increasingly rounded as the biofilm towers matured over 5 days. The role of gliding motility in biofilm formation was addressed by analyzing differences in biofilm architecture in non-motile mutant II-3R and hypermotile mutant prpC-and by using time-lapse microcinematography to measure flux of cells around biofilm towers. There were no major differences in biofilm architecture between WT and motility mutants, with perhaps a slight tendency for the prpC- cells to spread outside towers during early stages of biofilm formation. Consistent with an insignificant role of motility in biofilm development, flux of cells near towers, which was low, was dominated by exit of cells. Immunofluorescence microscopy revealed that motility-associated attachment organelle (AO) proteins exhibited no discernable changes in localization to foci over time, but immunoblotting identified a decrease in steady-state levels of protein P200, which is required for normal gliding speed, as the WT culture aged. Non-adherent strain II-3 and non-motile strain II-3R also exhibited a steady decrease in P200 steady-state levels, suggesting that the decrease in P200 levels was not a response to changes in gliding behavior during maturation. We conclude that M. pneumoniae cells undergo morphological changes as biofilms mature, motility plays no major role in biofilm development, and P200 loss might be related to maturation of cells. This study helps to characterize potential therapeutic targets for M. pneumoniae infections.

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution.

Although mycoplasmas have small genomes, many of them, including the HIV-associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) that is used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas, as well as compositional and ultrastructural differences among these AOs, suggests that AOs have arisen several times through convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO with several forms of microscopy and biochemical analysis, to determine whether the M. penetrans AO was constructed at the molecular level on principles similar to those of other mycoplasmas, such as Mycoplasma pneumoniae and Mycoplasma mobile We found that the M. penetrans AO interior was generally dissimilar from that of other mycoplasmas, in that it exhibited considerable heterogeneity in size and shape, suggesting a gel-like nature. In contrast, several of the 12 potential protein components identified by mass spectrometry of M. penetrans detergent-insoluble proteins shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, although not with M. mobile proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level, leading to the possibility that the less organized material in both M. pneumoniae and M. penetrans is the substance principally responsible for the organization and function of the AO.IMPORTANCEMycoplasma penetrans is a bacterium that infects HIV-positive patients and may contribute to the progression of AIDS. It attaches to host cells through a structure called an AO, but it is not clear how it builds this structure. Our research is significant not only because it identifies the novel protein components that make up the material within the AO that give it its structure but also because we find that the M. penetrans AO is organized unlike AOs from other mycoplasmas, suggesting that similar structures have evolved multiple times. From this work, we derive some basic principles by which mycoplasmas, and potentially all organisms, build structures at the subcellular level.

Cellular Microbiology of Mycoplasma canis.

Mycoplasma canis can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence of M. canis in brains of dogs with idiopathic meningoencephalitis prompted new in vitro studies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P < 0.01) among strains of M. canis isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (P < 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation with M. canis also decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (P < 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of M. canis polyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen, M. canis has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemical in vivo milieu.

Aerosol tracer testing in Boeing 767 and 777 aircraft to simulate exposure potential of infectious aerosol such as SARS-CoV-2.

The COVID-19 pandemic has reintroduced questions regarding the potential risk of SARS-CoV-2 exposure amongst passengers on an aircraft. Quantifying risk with computational fluid dynamics models or contact tracing methods alone is challenging, as experimental results for inflight biological aerosols is lacking. Using fluorescent aerosol tracers and real time optical sensors, coupled with DNA-tagged tracers for aerosol deposition, we executed ground and inflight testing on Boeing 767 and 777 airframes. Analysis here represents tracer particles released from a simulated infected passenger, in multiple rows and seats, to determine the exposure risk via penetration into breathing zones in that row and numerous rows ahead and behind the index case. We present here conclusions from 118 releases of fluorescent tracer particles, with 40+ Instantaneous Biological Analyzer and Collector sensors placed in passenger breathing zones for real-time measurement of simulated virus particle penetration. Results from both airframes showed a minimum reduction of 99.54% of 1 µm aerosols from the index source to the breathing zone of a typical passenger seated directly next to the source. An average 99.97 to 99.98% reduction was measured for the breathing zones tested in the 767 and 777, respectively. Contamination of surfaces from aerosol sources was minimal, and DNA-tagged 3 µm tracer aerosol collection techniques agreed with fluorescent methodologies.

Potential Molecular Targets for Narrow-Spectrum Agents to Combat Mycoplasma pneumoniae Infection and Disease.

As Mycoplasma pneumoniae macrolide resistance grows and spreads worldwide, it is becoming more important to develop new drugs to prevent infection or limit disease. Because other mycoplasma species have acquired resistance to other classes of antibiotics, it is reasonable to presume that M. pneumoniae can do the same, so switching to commonly used antibiotics like fluoroquinolones will not result in forms of therapy with long-term utility. Moreover, broad-spectrum antibiotics can have serious consequences for the patient, as these drugs may have severe impacts on the natural microbiota of the individual, compromising the health of the patient either short-term or long-term. Therefore, developing narrow-spectrum antibiotics that effectively target only M. pneumoniae and no more than a small portion of the microbiota is likely to yield impactful, positive results that can be used perhaps indefinitely to combat M. pneumoniae. Development of these agents requires a deep understanding of the basic biology of M. pneumoniae, in many areas deeper than what is currently known. In this review, we discuss potential targets for new, narrow-spectrum agents and both the positive and negative aspects of selecting these targets, which include toxic molecules, metabolic pathways, and attachment and motility. By gathering this information together, we anticipate that it will be easier for researchers to evaluate topics of priority for study of M. pneumoniae.