David Pezoa

Objective: The Type VI Secretion System (T6SS) is a contractile apparatus made of several proteins playing a significant role in the fitness and virulence of many Gram-negative bacteria. The SPI-19 T6SS gene cluster is a virulence factor of Salmonella Dublin contributing to host colonization and antibacterial activity. Previously, we demonstrated that SED_RS06335 (an Rhs protein with a C-terminal M91 domain) is responsible for antibacterial activity of the T6SS _SPI−19 ; however, the role of the M91 domain in this phenotype is unknown. Thus, the objective of this study is to provide experimental evidence that the antibacterial activity of SED_RS06335 effector is attributable to its C-terminal M91 domain. Results: Here, we determine through interbacterial competition and heterologous expression assays that M91 domain of SED_RS06335 displays antibacterial activity. Furthermore, a three-dimensional structural model of M91 domain revealed a high similarity with the metallopeptidase neurotoxin type A (BotA) from Clostridium botulinum . Interestingly, our results suggest that the M91 domain is responsible for the antibacterial activity of the SED_RS06335 effector in Salmonella Dublin.

The type VI secretion system (T6SS) is a contact-dependent, contractile multiprotein complex widely distributed among Gram-negative bacteria. It mediates the translocation of effector proteins into bacterial competitors and eukaryotic host cells, promoting environmental fitness and contributing to virulence. In Salmonella, five pathogenicity islands encoding T6SSs (SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22) have been described, along with an expanding repertoire of associated effector proteins. However, their global diversity and distribution remain incompletely resolved due to limited genomic sampling. To address this, we analyzed a curated dataset of 490 Salmonella genomes representing 45 serotypes. T6SS regions were identified using SecreT6, revealing that SPI-6 is widely distributed, whereas SPI-19, SPI-20, and SPI-21 are restricted to a subset of serotypes. SPI-20 and SPI-21 were exclusively found in S. enterica subsp. arizonae and diarizonae, while SPI-22 was absent from all analyzed genomes. All open reading frames within T6SS clusters were then analyzed for effector prediction and functional annotation. This approach recovered 32 out of 45 previously described T6SS effectors and identified several novel candidates. These included a cytidine deaminase with predicted DNase activity in SPI-6: two candidate nuclease effectors in SPI-19 with DNase and RNase activities, and four putative effectors in SPI-21, including enzymes with predicted peptidoglycan hydrolase activity, a potential inhibitor of eukaryotic ATPases, and a membrane pore-forming toxin. Additionally, a putative phospholipase effector was identified within a VgrG-associated genomic island in a subset of S. enterica subsp. diarizonae isolates. Collectively, these findings expand the known repertoire of Salmonella T6SS effector proteins and highlight their functional diversity.

The type VI secretion system (T6SS) is a contact-dependent multiprotein apparatus widely distributed in Gram-negative bacteria that contributes to interbacterial competition and pathogenesis via a contractile mechanism. In Salmonella , five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. One of the most studied and widely distributed T6SS corresponds to that encoded in SPI-6 (T6SS _SPI-6 ), which contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, there is still limited information available regarding the total number of effector proteins encoded within SPI-6 of different Salmonella enterica serotypes. In the present study, we characterized the SPI-6 T6SS gene cluster encoded in Salmonella enterica subspecies enterica serotype Tennessee ( S . Tennessee), a pathogen frequently associated with foodborne gastrointestinal outbreaks. Interbacterial competition assays demonstrated that T6SS _SPI-6 of S. Tennessee displays antibacterial activity. Additionally, we performed comparative genomic and bioinformatic analyses and identified an antibacterial Effector/Immunity protein (E/I) module encoding a putative effector with DNase activity (RhsA-HNHc) and its cognate immunity protein within the variable region 3 (VR3) of the SPI-6 T6SS gene cluster. Interbacterial competition assays confirmed the antibacterial activity of this novel E/I pair. In addition, heterologous expression assays showed that induction of the RhsA-HNHc effector led to significant E. coli growth inhibition, while co-expression with its putative immunity protein fully restored bacterial growth, thus demonstrating protection against toxicity. Finally, a nuclease activity assay demonstrated that RhsA-HNHc possesses DNase activity. Altogether, this study expands the experimentally validated SPI-6 T6SS effector repertoire beyond well-studied Salmonella serotypes, providing the first functional characterization of a DNase-type Rhs effector in S . Tennessee.

The type VI secretion system (T6SS) is a contact-dependent multiprotein apparatus that contributes to interbacterial competition and pathogenesis in many Gram-negative bacteria. Salmonella harbors five T6SS gene clusters within the pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, differentially distributed among serotypes. Salmonella enterica subspecies arizonae ( S. arizonae ) is most frequently associated with reptiles but, in some circumstances, can cause disease in mammals, including humans. Notably, although it encodes both T6SS _SPI-20 and T6SS _SPI-21 , no report to date has demonstrated the antibacterial activity of either system. In addition, only two putative effector proteins have been previously predicted, though they have not been experimentally validated. In the present study, we demonstrate that both T6SS _SPI-20 and T6SS _SPI-21 contribute to interbacterial competition in S. arizonae when grown on McConkey agar plates, suggesting a role for bile in T6SS _SPI-20 and T6SS _SPI-21 activity. In addition, through bioinformatic analyses, interbacterial competition assays, and heterologous expression, we further characterize the antibacterial activity of a novel antibacterial E/I module (SARI_02625/SARI_02624, encoded in SPI-21) that, in addition to the previously identified evolved VgrG protein SARI_02603 and VgrG2b homolog SARI_02727, contribute to T6SS-dependent antibacterial competition in S. arizonae .

Carbapenemase-mediated resistance, driven by enzymes that inactivate last resort carbapenem antibiotics, has emerged as a critical global health threat. To delineate its true scope, we conducted an exhaustive review of the past five years’ literature, focusing on the five principal carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) in human clinical isolates. All relevant studies were synthesized into a unified database, which served as the basis for generating individual world maps for each enzyme and a composite map illustrating their co-occurrence. These visual tools illuminate the uneven yet interconnected nature of carbapenemase dissemination, underscoring the value of spatially resolved data in combating antimicrobial resistance. Our findings reveal distinct regional patterns: KPC dominates much of the Americas and Western Europe; NDM is most burdensome in the Indian subcontinent and North Africa; VIM remains concentrated in Southern Europe; IMP is endemic in Japan and parts of China; and OXA-48-like enzymes prevail around the Mediterranean and in the Middle East. These disparities reflect local genetic transfer dynamics, via plasmids, integrons, and other mobile elements, as well as variations in antibiotic use practices and surveillance capacity. Accurate, up-to-date distribution maps are therefore indispensable for tailoring surveillance strategies, optimizing antimicrobial stewardship, and directing research funding, all of which are vital for slowing the spread of these formidable resistance determinants. La resistencia mediada por carbapenemasas, enzimas capaces de neutralizar los carbapenémicos, antibióticos considerados de última línea terapéutica, constituye una amenaza sanitaria de alcance global. Para caracterizar su verdadero alcance, realizamos una búsqueda exhaustiva de la literatura científica de los últimos cinco años, enfocada en la detección de las cinco carbapenemasas principales (KPC, NDM, VIM, IMP y OXA-48-like) en aislamientos humanos, y se consolidó la información obtenida en una base de datos unificada. A partir de ella se generaron mapas de distribución individual para cada enzima y un mapa compuesto que refleja la co-ocurrencia de todas. Los resultados revelan patrones geográficos distintivos: KPC domina en gran parte de América y Europa occidental; NDM presenta su mayor carga en el subcontinente indio y el norte de África; VIM se concentra en el sur de Europa; IMP mantiene su endemia en Japón y China; y OXA-48-like es especialmente prevalente en la región del Mediterráneo y Oriente Medio. Estas diferencias responden tanto a dinámicas locales de transferencia genética, plásmidos, integrones y elementos móviles, como a prácticas de uso de antibióticos y niveles de vigilancia microbiológica. Disponer de mapas precisos y actualizados resulta crucial para adaptar las políticas de vigilancia, optimizar estrategias de control y orientar la asignación de recursos y esfuerzos de investigación, con el fin de contener la diseminación de estas enzimas y preservar la eficacia de los tratamientos de última línea.

The Type VI Secretion System (T6SS) is a critical fitness and virulence factor of many Gram-negative bacteria. Five T6SS gene clusters have been described in Salmonella , each one encoded within different pathogenicity islands (i.e., SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22). The events of gain and loss of these T6SS gene clusters have contributed to shape the genome evolution of different Salmonella serotypes. In addition, the differential distribution of T6SS and the ever-increasing repertoire of predicted effector proteins is likely to play an important role in the environmental fitness and pathogenic potential of different Salmonella serotypes. This review summarizes the current knowledge on the role played by T6SS in Salmonella biology, highlighting the major milestones in the field over the past two decades. We discuss the expanding repertoire of T6SS effector proteins identified to date and examine the current understanding of mechanisms controlling T6SS expression in Salmonella , focusing on host-derived cues and regulators involved. Finally, we provide a critical analysis of conflicting reports and suggest future directions for the research in the field. A better understanding of these processes could expand our knowledge of Salmonella biology, and the mechanisms behind how this versatile secretion system enables Salmonella to thrive in competitive microbial environments and contribute to host colonization.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 new putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella . The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 novel putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica . The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

Salmonella genus harbors five Type VI Secretion System (T6SS) gene clusters. The T6SS encoded in SPI-6 (T6SS_SPI-6) contributes to Salmonella Typhimurium colonization of chickens and mice, while the T6SS encoded in SPI-19 (T6SS_SPI-19) of Salmonella Gallinarum contributes to chicken colonization. Interestingly, the T6SS_SPI-19 of Salmonella Gallinarum complemented the defect in chicken colonization of a Salmonella Typhimurium strain that lacks the T6SS_SPI-6, suggesting that both T6SSs are interchangeable. Here we show that the transfer of Salmonella Gallinarum T6SS_SPI-19 complemented the defect in mice colonization of a Salmonella Typhimurium ΔT6SS_SPI-6 strain, indicating that both T6SSs are functionally redundant during host colonization.