RESUMO
Shigella flexneri can synthesize polysaccharide chains having complex sugars and a regulated number of repeating units. S. flexneri lipopolysaccharide O antigen (Oag) is synthesized by the Wzy-dependent pathway, which is the most common pathway used in bacteria for polysaccharide synthesis. The inner membrane protein WzyB polymerizes the Oag repeat units into chains, while the polysaccharide copolymerases WzzB and WzzpHS2 determine the average number of repeat units or "the modal length," termed short type and very long type. Our data show for the first time a direct interaction between WzyB and WzzpHS2, with and without the use of the chemical cross-linker dithiobis (succinimidyl propionate) (DSP). Additionally, mutations generated via random and site-directed mutagenesis identify a region of WzyB that caused diminished function and significantly decreased very long Oag chain polymerization, and that affected the aforementioned interaction. These results provide insight into the mechanisms underlying the regulation of Oag biosynthesis. IMPORTANCE Complex polysaccharide chains are synthesized by bacteria, usually at a regulated number of repeating units, which has broad implications for bacterial pathogenesis. One example is the O antigen (Oag) component of lipopolysaccharide that is predominantly synthesized by the Wzy-dependent pathway. Our findings show for the first time a direct physical interaction between WzyB and WzzpHS2. Additionally, a set of Wzy mutant constructs were generated, revealing a proposed active site/switch region involved in the activity of WzyB and the physical interaction with WzzpHS2. Combined, these findings further understanding of the Wzy-dependent pathway. The identification of a novel interaction with the polysaccharide copolymerase WzzpHS2 and the region of WzyB that is involved in this aforementioned interaction and its impact on WzyB Oag synthesis activity have significant implication for the prevention/treatment of bacterial diseases and discovery of novel biotechnologies.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano , Mutagênese , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Shigella flexneri/genéticaRESUMO
Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.
Assuntos
Bacteriófagos/metabolismo , Disenteria Bacilar/microbiologia , Antígenos O/metabolismo , Receptores Virais/metabolismo , Shigella flexneri/metabolismo , Shigella flexneri/virologia , Acetilação , Bacteriófagos/genética , Células HeLa , Humanos , Antígenos O/genética , Receptores Virais/genética , Sorogrupo , Shigella flexneri/classificação , Shigella flexneri/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The Shigella flexneri IcsA (VirG) protein is a polarly distributed autotransporter protein. IcsA functions as a virulence factor by interacting with the host actin regulatory protein N-WASP, which in turn activates the Arp2/3 complex, initiating actin polymerization. Formation of F-actin comet tails allows bacterial cell-to-cell spreading. Although various accessory proteins such as periplasmic chaperones and the ß-barrel assembly machine (BAM) complex have been shown to be involved in the export of IcsA, the IcsA translocation mechanism remains to be fully elucidated. A putative autochaperone (AC) region (amino acids 634-735) located at the C-terminal end of the IcsA passenger domain, which forms part of the self-associating autotransporter (SAAT) domain, has been suggested to be required for IcsA biogenesis, as well as for N-WASP recruitment, based on mutagenesis studies. IcsA(i) proteins with linker insertion mutations within the AC region have a significant reduction in production and are defective in N-WASP recruitment when expressed in smooth LPS (S-LPS) S. flexneri. In this study, we have found that the LPS O antigen plays a role in IcsA(i) production based on the use of an rmlD (rfbD) mutant having rough LPS (R-LPS) and a novel assay in which O antigen is depleted using tunicamycin treatment and then regenerated. In addition, we have identified a new N-WASP binding/interaction site within the IcsA AC region.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Mutação , Antígenos O/biossíntese , Shigella flexneri/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/metabolismo , Disenteria Bacilar/microbiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Shigella flexneri/química , Shigella flexneri/genética , Fatores de Transcrição/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Shigella flexneri serotype 2a2 (II:9;10) is the most prevalent strain in causing bacillary dysentery in developing countries. Chemical modifications such as glucosylation, O-acetylation, and phosphoethanolamine modifications of lipopolysaccharide (LPS) O antigen (Oag) contribute to the emergence of various serotypes. Sf6 is a Shigella-specific bacteriophage that infects only a limited range of S. flexneri serotypes [X, Y]. LPS Oag is the primary receptor for bacteriophage Sf6 where it uses its tailspike protein (TSP) in binding and hydrolysing LPS Oags. Sf6TSP has recently been shown to be capable of hydrolysing the LPS Oag of Type II strains, albeit modestly. Phage therapy has regained attention in recent years as an alternative therapeutic approach. Therefore, this study aimed to expand the host range of Sf6 to the prevalent S. flexneri serotype 2a2 strain. We discovered a new lytic Sf6 host range mutant that is capable of infecting S. flexneri serotype 2a2 and identified residues in Sf6TSP that may potentially be involved in binding and hydrolysing serotype 2a2 LPS Oag. This work increased the limited Shigella-specific bacteriophage collection and may be useful in the future for phage therapy and/or biocontrolling of S. flexneri in contaminated food and water.
Assuntos
Bacteriófagos , Disenteria Bacilar , Bacteriófagos/genética , Especificidade de Hospedeiro , Humanos , Antígenos O/química , Sorogrupo , Shigella flexneri/genéticaRESUMO
The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials. One such biomaterial is bacterial cellulose, which is a strong and ultrapure natural polymer used in tissue engineering scaffolds, wound dressings, electronics, food additives, and other products. However, despite the potential of Acetobacteraceae in biotechnology, there has been considerably little effort to fundamentally reprogram the bacteria for enhanced performance. One limiting factor is the lack of a well-characterized, comprehensive toolkit to control expression of genes in biosynthetic pathways and regulatory networks to optimize production and cell viability. Here, we address this shortcoming by building an expanded genetic toolkit for synthetic biology applications in Acetobacteraceae. We characterized the performance of multiple natural and synthetic promoters, ribosome binding sites, terminators, and degradation tags in three different strains, namely, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 53582, and Komagataeibacter rhaeticus iGEM. Our quantitative data revealed strain-specific and common design rules for the precise control of gene expression in these industrially relevant bacterial species. We further applied our tools to synthesize a biodegradable cellulose-chitin copolymer, adjust the structure of the cellulose film produced, and implement CRISPR interference for ready down-regulation of gene expression. Collectively, our genetic parts will enable the efficient engineering of Acetobacteraceae bacteria for the biomanufacturing of cellulose-based materials and other commercially valuable products.
Assuntos
Acetobacteraceae/genética , Expressão Gênica/genética , Bebidas/microbiologia , Materiais Biocompatíveis/metabolismo , Vias Biossintéticas/genética , Biotecnologia/métodos , Celulose/genética , Quitina/genética , Alimentos , Biologia Sintética/métodos , Engenharia Tecidual/métodosRESUMO
Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri. We first completed a tyrosine phosphoproteome, identifying 905 unique tyrosine phosphorylation sites on at least 573 proteins (approximately 15% of all proteins). This is the most tyrosine-phosphorylated sites and proteins in a single bacterium identified to date, substantially more than the level seen in eukaryotic cells. Most had not previously been identified and included proteins encoded by the virulence plasmid, which is essential for S. flexneri to invade cells and cause disease. In order to investigate the function of these phosphorylation sites in important virulence factors, phosphomimetic and ablative mutations were constructed in the type 3 secretion system ATPase Spa47 and the master virulence regulator VirB. This revealed that tyrosine residues phosphorylated in our study are critical for Spa47 and VirB activity, and tyrosine phosphorylation likely regulates their functional activity and subsequently the virulence of this major human pathogen. This study suggests that tyrosine phosphorylation plays a critical role in regulating a wide variety of virulence factors in the human pathogen S. flexneri and serves as a base for future studies defining its complete role.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Tirosina/metabolismo , Células HeLa , Humanos , Fosforilação , Proteoma/análise , VirulênciaRESUMO
The Shigella flexneri autotransporter protein IcsA is essential for intra- and intercellular spread, and icsA mutants are attenuated in several models. However, the pathogenic significance of the outer membrane protease IcsP, which orchestrates the polar distribution of IcsA on the bacterial surface, remains unclear. To further examine this point, we constructed icsP mutants in the two most commonly studied S. flexneri strains and evaluated their in vitro and in vivo performance relative to wild type. Both icsP mutants showed aberrant surface distribution of IcsA, but the in vitro consequences depended upon the cell line being used to assess bacterial motility and plaque formation. Evaluating the behaviour of the mutants in two mouse models suggested functional expression of icsP might limit bacterial persistence and the associated inflammation in host tissues, consistent with the findings in one of the three cell lines used.
Assuntos
Proteínas de Bactérias/metabolismo , Shigella flexneri/genética , Shigella flexneri/patogenicidade , Actinas/genética , Aminoglicosídeos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Células CACO-2 , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/microbiologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fatores de Transcrição/metabolismoRESUMO
The Shigella flexneri IcsA (VirG) protein is a polarly distributed outer membrane protein that is a fundamental virulence factor which interacts with neural Wiskott-Aldrich syndrome protein (N-WASP). The activated N-WASP then activates the Arp2/3 complex which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allows bacterial cell-to-cell spreading. In a previous study, IcsA was found to have three N-WASP interacting regions (IRs): IR I (aa 185-312), IR II (aa 330-382) and IR III (aa 508-730). The aim of this study was to more clearly define N-WASP interacting regions II and III by site-directed mutagenesis of specific amino acids. Mutant IcsA proteins were expressed in both smooth lipopolysaccharide (S-LPS) and rough LPS (R-LPS) S. flexneri strains and characterised for IcsA production level, N-WASP recruitment and F-actin comet tail formation. We have successfully identified new amino acids involved in N-WASP recruitment within different N-WASP interacting regions, and report for the first time using co-expression of mutant IcsA proteins, that N-WASP activation involves interactions with different regions on different IcsA molecules as shown by Arp3 recruitment. In addition, our findings suggest that autochaperone (AC) mutant protein production was not rescued by another AC region provided in trans, differing to that reported for two other autotransporters, PrtS and BrkA autotransporters.