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Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)

Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.

Importance: As a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Pathogenic serotypes of Vibrio cholerae, transmitted through contaminated water and food, are responsible for outbreaks of cholera, an acute diarrheal disease. While the cholera toxin is the primary virulence factor, V. cholerae also expresses other virulence factors, such as the tripartite toxin MakABE that is secreted via the bacterial flagellum. These three proteins are co-expressed with two accessory proteins, MakC and MakD, whose functions remain unknown. Here, we present the crystal structures of MakC and MakD, revealing that they are similar in both sequence and structure but lack other close structural relatives. Our study further investigates the roles of MakC and MakD, focusing on their impact on the expression and secretion of the components of the MakABE tripartite toxin. Through deletion mutant analysis, we found that individual deletions of makC or makD do not significantly affect MakA expression or secretion. However, the deletion of both makC and makD impairs the expression of MakB, which is directly downstream, and decreases the expression of MakE, which is separated from makCD by two genes. Conversely, MakA, encoded by the makA gene located between makB and makE, is expressed normally but its secretion is impaired. Additionally, our findings indicate that MakC, in contrast to MakD, exhibits strong interactions with other proteins. Furthermore, both MakC and MakD were observed to be localized within the cytosol of the bacterial cell. This study provides new insights into the regulatory mechanisms affecting the Mak protein family in V. cholerae and highlights the complex interplay between gene proximity and protein expression.

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

Recently, we demonstrated that a novel bacterial cytotoxin, the protein MakA which is released by Vibrio cholerae, is a virulence factor, causing killing of Caenorhabditis elegans when the worms are grazing on the bacteria. Studies with mammalian cell cultures in vitro indicated that MakA could affect eukaryotic cell signalling pathways involved in lipid biosynthesis. MakA treatment of colon cancer cells in vitro caused inhibition of growth and loss of cell viability. These findings prompted us to investigate possible signalling pathways that could be targets of the MakA-mediated inhibition of tumour cell proliferation. Initial in vivo studies with MakA producing V. cholerae and C. elegans suggested that the MakA protein might target the PIP5K1α phospholipid-signalling pathway in the worms. Intriguingly, MakA was then found to inhibit the PIP5K1α lipid-signalling pathway in cancer cells, resulting in a decrease in PIP5K1α and pAkt expression. Further analyses revealed that MakA inhibited cyclin-dependent kinase 1 (CDK1) and induced p27 expression, resulting in G2/M cell cycle arrest. Moreover, MakA induced downregulation of Ki67 and cyclin D1, which led to inhibition of cell proliferation. This is the first report about a bacterial protein that may target signalling involving the cancer cell lipid modulator PIP5K1α in colon cancer cells, implying an anti-cancer effect.

Efficient systemic pharmacological treatment of solid tumors is hampered by inadequate tumor concentration of cytostatics necessitating development of smart local drug delivery systems. To overcome this, we demonstrate that doxorubicin (DOX), a cornerstone drug used for osteosarcoma treatment, shows reversible accretion to hydroxyapatite (HA) of both nano (nHA) and micro (mHA) size. nHA particles functionalized with DOX get engulfed in the lysosome of osteosarcoma cells where the acidic microenvironment causes a disruption of the binding between DOX and HA. The released DOX then accumulates in the mitochondria causing cell starvation, reduced migration and apoptosis. The HA+DOX delivery system was also tested in-vivo on osteosarcoma bearing mice. Locally delivered DOX via the HA particles had a stronger tumor eradication effect compared to the controls as seen by PET-CT and immunohistochemical staining of proliferation and apoptosis markers. These results indicate that in addition to systemic chemotherapy, an adjuvant nHA could be used as a carrier for intracellular delivery of DOX for prevention of tumor recurrence after surgical resection in an osteosarcoma. Furthermore, we demonstrate that nHA particles are pivotal in this approach but a combination of nHA with mHA could increase the safety associated with particulate nanomaterials while maintaining similar therapeutic potential.

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.