Publications

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

JTD Keywords: Antibodies, Beta-lactamase, Cell, Enterica, Escherichia-coli, Expression, Genes, Infection, Intimin, Mous, Mutational analysis, Pcr, Salmonella, Var typhimurium

Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.

JTD Keywords: antimicrobial resistance, bacterial ig-like proteins, bacterial lg-like proteins, chromosomal genes, identification, inca/c, mutational analysis, plasmid conjugation, products, r-factors, resistance plasmids, salmonella-enterica, sequence, Antimicrobial resistance, Bacterial ig-like proteins, Escherichia-coli, Plasmid conjugation

The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer. The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer.

JTD Keywords: HGT, H-NS, StpA, Salmonella, SPI1

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence.

JTD Keywords: Salmonella, Gene regulation, Motility, Pathogenicity island, H-NS, HHA, STPA

A genetic screening for osmoregulated genes allowed us to identify the yfeR gene of Salmonella enterica serovar Typhimurium. The yfeR gene product encodes a novel LysR-type transcriptional regulator (LTTR), the expression of which decreases when external osmolarity increases. Out of the adjacent gene yfeH, YfeR modulates expression of several genes that may be required for optimal growth under low osmolarity conditions.

JTD Keywords: YfeR, Salmonella, LysR, Osmoregulation, LTTR

Horizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.

JTD Keywords: Hha, H-NS, HGT DNA, Enterobacteria, Nucleoid-associated proteins, Enterica serovar typhimurium, Histone-like protein, h-ns, Escherichia-coli, Yersinia-enterocolitica, Salmonella-enterica

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

JTD Keywords: 2A strain 2457T, Escherichia-Coli, Salmonella-Enterica, Protein, DNA, Expression, Binding, HHA, Shigella, Plasmid

The Streptococcus pyogenes genome harbors two clusters of class Ib ribonucleotide reductase genes, nrdHEF and nrdF*I*E*, and a second stand-alone nrdI gene, designated nrdI2. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in Escherichia coli. The nrdF*I*E* operon requires the presence of the nrdI* gene, and the nrdHEF operon gained activity upon cotranscription of the heterologous nrdI gene from Streptococcus pneumoniae, while neither nrdI* nor nrdI2 from S. pyogenes rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the nrdF*I*E* operon has been horizontally transferred to S. pyogenes from Mycoplasma spp.

JTD Keywords: Group-a streptococcus, Bacillus-subtilis genes, Escherichia-coli, Corynebacterium-ammoniagenes, Mycobacterium-tuberculosis, Expression analysis, Genome sequence, Small-subunit, Salmonella-typhimurium, Iron center