Pilus production in Acinetobacter baumannii is growth phase dependent and essential for natural transformation

Acinetobacter baumannii is a severe threat to human health as a frequently multidrug-resistant hospital-acquired pathogen. Part of the danger from this bacterium comes from its genome plasticity and ability to evolve quickly by taking up and recombining external DNA into its own genome in a process called natural competence for transformation. This mode of horizontal gene transfer is one of the major ways pathogens can acquire new antimicrobial resistances and toxic traits. Because these processes in A. baumannii are not well studied, we herein characterized new aspects of natural transformability in this species that include the species’ competence window. We uncovered a strong correlation with a growth–phase-dependent synthesis of a type IV pilus (TFP), which constitutes the central part of competence-induced DNA-uptake machinery. We used bacterial genetics and microscopy to demonstrate that the TFP is essential for the natural transformability and surface motility of A. baumannii, whereas pilus-unrelated proteins of the DNA-uptake complex do not impact the motility phenotype. Furthermore, TFP biogenesis and assembly is subject to input from two regulatory systems that are homologous to Pseudomonas aeruginosa, namely the PilSR two-component system and the Pil-Chp chemosensory system. We demonstrated that these systems not only impact the piliation status of cells but also their ability to take up DNA for transformation. Importantly, we report on discrepancies between TFP biogenesis and natural transformability within the same genus by comparing A. baumannii to data reported for A. baylyi, the latter of which served for decades as a model for natural competence.

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

Horizontal gene transfer (HGT) has a major impact on bacterial evolution, leading to acquisition or deletion of genes and gene clusters, including those encoding antibiotic resistances and virulence factors. HGT therefore contributes to pathogen emergence, which can have a major impact on human health. As a mode of HGT, natural competence for transformation allows bacteria to directly acquire DNA from the environment and to integrate parts of this genetic material within their genomes. The human pathogen Vibrio cholerae is naturally competent when it grows in association with the chitinous exoskeletons of zooplankton. Under this condition, the competence state is initiated by the regulatory protein TfoX, which co-induces a molecular killing device known as the type VI secretion system (T6SS). The co-regulation of the T6SS and competence results in the lysis of non-immune neighboring bacteria and the subsequent acquisition of their DNA, which fosters HGT. In V. cholerae, the T6SS is also activated by the TfoX homologue protein, named TfoY. In this study, we aimed at studying the regulatory pathways and the co-regulation of interbacterial predation and DNA uptake in order to provide new insights into the environmental lifestyle of V. cholerae and other vibrios. We first investigated the conserved function of TfoX- and TfoY in V. fischeri, V. alginolyticus, and V. parahaemolyticus using diverse methods such as qRT-PCR, interbacterial killing assays, and motility assessments. We demonstrated that TfoX-mediated T6SS and competence induction and TfoY-induced motility are conserved phenotypes in the tested Vibrio species, whereas the TfoY-mediated T6SS regulation varied. These variations might reflect diverse defense mechanisms, as these different species have adapted to cope with their environmental niches. Next, we tested the outcome of the TfoX-mediated co-regulation of the T6SS and competence in V. cholerae. Under conditions mimicking the bacteriumâs natural habitat, we showed for the first time the extent of DNA that was integrated on V. choleraeâs genome after T6SS-mediated interbacterial predation. Indeed, we demonstrated that T6SS-dependent bacterial predation not only increased the transformation rates but also fostered the exchange of multiple and huge fragments of DNA. We also showed the necessity of an exquisite co-regulation between the T6SS, the competence-related DNA uptake machinery, and the competence-repressed nuclease. Finally, we disclosed that prey-released DNA is not a private good of the attacking bacterium but also accessible to other surrounding cells that have likewise entered the competence state. Altogether, our findings shed light on the conservation of the TfoX- and TfoY-driven phenotypes in several Vibrio species. We also contributed to a better understanding of the interplay between neighbor predation and DNA uptake and the consequences that this interplay has on genome plasticity.

EPFL2019

Natural competence of the pathogen Acinetobacter baumannii: an elusive phenomenon

Nina Vesel

Natural competence for transformation is an important driver of horizontal DNA exchange between different organisms. This can result in accumulation of dangerous genetic features, such as antibiotic resistance genes, in a single organism. One example of an organism that frequently acquires antibiotic resistance genes is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that has recently become problematic in hospital settings. Even though natural competence for transformation was demonstrated for some A. baumannii isolates, its competence regulon, the exact mechanism of DNA uptake, and the contribution of transformation to the acquisition of antibiotic resistance genes were largely unexplored at the beginning of this thesis. The aim of this thesis was therefore to better characterize optimal conditions of competence, the underlying competence regulon, and the necessary set of machinery proteins. Additionally, we aimed to identify the limiting factors that oppose transformation-mediated exchange of DNA in a small selection of strains. Here, we first addressed the general aspects of natural transformation, such as the competence window and the DNA uptake machinery. Our results showed that transformation is growth phase-dependent in A. baumannii and correlated with type IV pili (T4P) production. We demonstrated that T4P are essential for transformation and surface-associated motility but are only produced in a subfraction of the bacterial population. Furthermore, T4P production and assembly were under control of the PilSR two component system and the chemotaxis-like Pil-Chp system, respectively. These two regulatory systems were essential for competence development in A. baumannii, which is in contrast to what was observed for its close relative A. baylyi. We also investigated the reasons for non-transformability of certain A. baumannii isolates. We showed that comM interruption as well as the diversity of PilA protein sequences did not explain the strains' non-transformability. Instead, decreased expression of certain pilus genes was associated to the absence of transformability, which was also reflected in the protein level of the major component of T4P - PilA. Since the previously identified regulators could not explain the reduced transcript levels of the respective pilus genes, a transposon sequencing screen was performed to identify novel transformation-relevant genes. By comparison of transcriptional profiles of such genes between the transformable and non-transformable strains, we identified possible candidates that might explain non-transformability. Lastly, we explored whether the epigenome can influence A. baumannii's transformability. Our results showed that the source of transforming DNA has a significant effect on transformation of certain, yet not all tested strains. Specifically, for strain A118 the transformation levels were significantly decreased when non-self DNA was used as the donor DNA. Consequently, we identified a A118-specific restriction modification (RM) system that methylated specific DNA sequences in this strain and fostered the discrimination of self versus foreign DNA. Collectively, the findings of this thesis deciphered several important aspects of natural transformation in A. baumannii, which might ultimately help to better understand the spread of antibiotic resistance genes in this organism.

EPFL2022

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014