The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani.

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

Lon Protease Is Important for Growth Under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani.

The Lon protein is a protease implicated in the virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria includes important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.

The Role of Proteases in the Virulence of Plant Pathogenic Bacteria.

A pathogenic lifestyle is inextricably linked with the constant necessity of facing various challenges exerted by the external environment (both within and outside the host). To successfully colonize the host and establish infection, pathogens have evolved sophisticated systems to combat the host defense mechanisms and also to be able to withstand adverse environmental conditions. Proteases, as crucial components of these systems, are involved in a variety of processes associated with infection. In phytopathogenic bacteria, they play important regulatory roles and modulate the expression and functioning of various virulence factors. Secretory proteases directly help avoid recognition by the plant immune systems, and contribute to the deactivation of the defense response pathways. Finally, proteases are important components of protein quality control systems, and thus enable maintaining homeostasis in stressed bacterial cells. In this review, we discuss the known protease functions and protease-regulated signaling processes associated with virulence of plant pathogenic bacteria.

The Extracellular Bacterial HtrA Proteins as Potential Therapeutic Targets and Vaccine Candidates.

BACKGROUND: An increasing resistance of bacteria to the commonly used antimicrobials forces to search for alternative or supportive ways to cure infections. Targeting virulence factors is one of such approaches. The bacterial HtrA proteins are strongly involved in virulence and the lack of functional HtrA in many cases impairs invasiveness of pathogens. HtrAs act by protecting the cells under stressful conditions as well as they take direct part in invasion of the host. The latter function is played predominantly by the recently identified extracellular fraction of HtrA. This review aims to evaluate HtrAs as therapeutic targets, including design of chemical inhibitors and vaccines. METHODS: We undertook a thorough search of bibliographic databases for peer-reviewed scientific literature. RESULTS: One hundred and sixty-four papers were included in the review. First, we briefly summarized key structural and functional properties of known HtrA proteins with the special focus on the extracellular HtrA fraction. Then we provided an overview of efforts and advancements to target HtrAs of pathogenic bacteria as a promising antimicrobial therapy. In some cases, encouraging results were obtained and application of HtrAspecific inhibitors protected tissues from damage and killed bacteria. Also promising reports concerning the use of HtrA as a protective antigen in several disease models have recently been published. CONCLUSION: The findings of this review suggest that the exported HtrA proteins are very attractive therapeutic targets due to their accessibility, significance in virulence and immunogenicity. However, further extensive studies are still needed to develop a safe antimicrobial treatment.