Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea in more than 100 million people worldwide every year. The bacteria replicate in the reproductive tract by evading innate and adaptive immunity. In the absence of effective vaccines and the rise of antibiotic resistance, understanding the molecular interactions between innate immune cells and N. gonorrhoeae may lead to new strategies to combat bacterial growth and the symptoms of gonorrhoea. It has long been known that the N. gonorrhoeae porin, PorB, promotes bacterial survival but also targets host mitochondria in infections. The mechanism by which PorB traffics form the bacterial outer membrane to host mitochondria remains unclear. Here, we utilized proteomics and super-resolution microscopy to show that N. gonorrhoeae secretes PorB via outer membrane vesicles. These vesicles are taken up by macrophages and deliver PorB to mitochondria. Macrophages treated with N. gonorrhoeae vesicles contained damaged mitochondria and active caspase-3. A caspase inhibitor prevented apoptosis of macrophages treated with N. gonorrhoeae vesicles. This suggests that N. gonorrhoeae secretes membrane vesicles, which are readily detectable in gonorrhoea patients, to target macrophages and to promote infections.

Significance: Staphylococcus aureus is one of the most significant human bacterial pathogens that has the capacity to cause serious infections and become highly resistant to antibiotics. In this study, we identified a metabolic adaptation mechanism used by S. aureus to simultaneously circumvent killing by one of the last-line antistaphylococcal antibiotics, daptomycin, and attack from host innate immune cells. This process led to enhanced bacterial survival and was mediated by a change in bacterial membrane phospholipid composition sufficient to impair daptomycin membrane penetration and significantly affect neutrophil chemotactic responses. These results highlight the importance of bacterial membrane lipid adaptation in bacterial pathogenesis and provide crucial insights into potentially novel therapeutic targeting.

SUMMARY: Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr ). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. Mitochondrial carrier proteins evolved during endosymbiosis to transport substrates across the mitochondrial inner membrane. As such the proteins are associated exclusively with eukaryotic organisms. Despite this, we identified putative mitochondrial carrier proteins in the genomes of different intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of Legionnaire's disease. We named the mitochondrial carrier protein from L. pneumophila LncP and determined that the protein is translocated into host cells during infection by the bacterial Dot/Icm type IV secretion system. From there, LncP accesses the classical mitochondrial import pathway and is incorporated into the mitochondrial inner membrane as an integral membrane protein. Remarkably, LncP crosses five biological membranes to reach its final location. Biochemically, LncP is a unidirectional nucleotide transporter similar to Aac1 in yeast. Although not essential for intracellular replication, the high carriage rate of lncP among isolates of L. pneumophila suggests that the ability of the pathogen to manipulate mitochondrial ATP transport assists survival of the bacteria in an intracellular environment.

A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy–infrared (AFM–IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in Staphylococcus aureus using AFM to demonstrate the division of the cell and AFM–IR to record spectra showing the thickening of the septum . This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from S. aureus and Escherichia coli containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM–IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor in vivo molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.

A shift in the Western diet since the industrial revolution has resulted in a dramatic increase in the consumption of omega-6 fatty acids, with a concurrent decrease in the consumption of omega-3 fatty acids. This decrease in omega-3 fatty acid consumption has been associated with significant disease burden, including increased susceptibility to infectious diseases. Here we provide evidence that DHA, an omega-3 fatty acid, has superior antimicrobial effects upon the highly drug-resistant pathogen Acinetobacter baumannii , thereby providing insights into one of the potential health benefits of omega-3 fatty acids. The identification and characterization of two novel bacterial membrane protective mechanisms against host fatty acids provide important insights into A. baumannii adaptation during disease. Furthermore, we describe a novel role for the major multidrug efflux system AdeIJK in A. baumannii membrane maintenance and lipid transport. This core function, beyond drug efflux, increases the appeal of AdeIJK as a therapeutic target.