MOLECULAR MECHANISMS OF VIRULENCE AND PATHOGENESIS OF PLANT PATHOGENIC BACTERIA.pptx

The ability of bacteria to cause disease is described in terms of the number of infecting bacteria, the route of entry into the body, the effects of host defense mechanisms, and intrinsic characteristics of the bacteria called virulence factors. Many virulence factors are so-called effector proteins that are injected into the host cells by specialized secretion apparati, such as the type three secretion system. Host-mediated pathogenesis is often important because the host can respond aggressively to infection with the result that host defense mechanisms do damage to host tissues while the infection is being countered

1. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE COLLEGE OF AGRICULTURE V. C. FARM MANDYA ASSIGNMENT TOPIC : Molecular mechanisms of pathogenesis and virulence of plant pathogeneic bacteria Reddy Kumar A V PAMM3005 Dept. of Plant Pathology

2. INTRODUCTION

3. BACTERIAL VIRULENCE The terms virulence and pathogenicity, which are often erroneously considered synonyms. Shurtleff and averre defined that pathogenicity is the ability of a pathogen to cause disease, whereas virulence is the degree of pathogenicity of a given pathogen. Most phytopathogens must evolve strategies to survive in different environmental conditions to invade and colonize their host known as virulence factors (Toth et al., 2003). Bacteria evade, overcome or suppress antimicrobial plant defences using these virulence factors, which elicit release of water and nutrients from host cells to colonize in the apoplast successfully.

4. Virulence factors: The molecules that assists the bacteria to colonize the host at the cellular level.

5. Production of extracellular polysaccharides Virulence factors Adhesion of bacteria to plant surfaces plant cell wall degrading enzymes Production of phytohormones Production of bacterial toxins Secretion system of bacteria

6. The first step in a successful colonization by pathogenic bacteria is their ability to maintain close proximity to a mucosal surface by adhesion. Adhesins are considered as biomolecules such as proteins and glycoproteins that mediate the binding of the bacteria to the host cell (Coa et al., 2001) . By irreversible binding with host cell receptors the appropriate organisms avoid being washed away by the fluids or being thrust out by ciliated cells. Bacterial cell adhesion to their host cell depends on the specific binding to carbohydrates presented at the cell surface which is mediated by adhesive organelles of bacteria, called fimbria. Adhesion of bacteria to plant surfaces

7. Plant pathogenic bacteria have evolved numerous sophisticated strategies for selective transport of proteins and nucleoproteins involved in the virulence across the cell membrane. Six major classes of systems implicated in the virulence have been identified in plant pathogenic bacteria from type I to type VI or T1SS to T6SS. In plant pathogenic Gram-negative bacteria, two major systems: * Single step process in which the secretion proteins are exported inner and outer membrane without periplasmic step. * The two steps process namely Sec and the Tat secretion system are first exported in periplasmic and then transported across the external membrane to the exterior of bacteria cell. Secretion system of bacteria

8. Secretion system of bacteria

9. Type I secretion system also known as the ATP binding cassette (ABC) transporters. ABC are involved in the export of various molecules from the cytosol to the external environment without periplasmic step ( Delepelaire et al., 2004). The type I secretion system have three different proteins that composed of continuous channel. ABC proteins transporters is specific outer membrane known as outer membrane protein (OMP) and also called as membrane fusion protein (MFP) which is connected to the inner membrane and spans the periplasmic space and extends to the outer membrane. Many proteins have great importance in pathogenesis are transferred by ABC secretion system in plant pathogenic bacteria including proteases, lipases or performing toxins. E.g. T1SS required bacteria are Erwinia amylovora and Dickeya chrysanthemi . Type 1 secretion system

10. T2SS uses a two-step process in which proteins transit the inner membrane in a Sec- or Tat-dependent process. The secreted proteins fold in the periplasmic space prior to passage through an outer membrane secretin pore ( Korotkov et al. 2012). T2SSs are used for the transport of many exoproteins, including proteases, lipases, and phosphatases. Examples of T2SS substrates include V. cholerae cholera toxin, enterotoxigenic E. coli (ETEC) LT toxin, and the P. aeruginosa virulence factors ExoA (exotoxin A), PlcH (hemolytic phospholipase C), LasA ( staphylolysin ), LasB ( pseudolysin elastase), PrpL (protease IV), AprA ( aeruginolysin ), ChiA (chitinase), and NanH (neuraminidase). Type 2 secretion system

11. Plant bacterial pathogens have evolved a strategy of delivering an array of effectors and toxins proteins directly into the cytoplasm of host cell. The type III secretion system apparatus is composed of more than of 20 proteins consisting of basal body spanning both inner and outer membrane of bacterial cells, and extra needle with the tip complex extending into the host cell. T3SS is encoded by hypersensitive response and pathogenicity ( hrp ) gene involved in the transfer of Avr proteins in the host cell (Galan and Collmer . 1999). Type 3 secretion system

12. Actin dynamics, mitogen-activated protein kinase (MAPK) signaling , and nuclear factor- κb ( nf-κb )-based inflammasome activation are modulated by enteropathogenic E. Coli (EPEC), enterohemorrhagic E. Coli (EHEC), P. Aeruginosa , and V. Cholerae T3SS effectors. Y. Pestis yop effectors also affect these signaling pathways as well as facilitating intracellular persistence within macrophage. The intracellular pathogens C. Trachomatis, S. Enterica , and S. Flexneri require T3SS effectors to invade and persist within the vacuolar system of host cells.

13. The type IV secretion system is present in both the Gram-negative and positive plant pathogenic bacteria ( Wallden et al., 2010). This translocation system that deploy the sec gene to transport the pathogenicity factors from the inner bacterial cell or into the extracellular environment or directly into the host cell. T4SS, which are structurally related to DNA conjugation systems, have the ability to transport DNA, DNA-protein complexes, and protein effectors across membranes. Type 4 secretion system

14. Agrobacterium tumefaciens that target the oncogenic dna -protein complex in the plant cell. N. Gonorrhoeae uses a T4SS to acquire virulence genes through horizontal gene transfer mechanisms. L. Pneumophila inject ~330 effector proteins that affect multiple host processes, including vesicle trafficking, autophagy, host protein synthesis, host inflammatory response, macrophage apoptosis, and host cell egress. H. Pylori uses the T4SS to insert effectors that modulate the host immune response. Examples for T4SS

15. Type 5 secretion system This type V secretion system is present in Gram-negative bacteria (Tseng et al., 2009). It is one of the simplest secretion pathway. The T5SS translocation system is dedicated to transfer a single specific polypeptide known as the passenger domain in two step process: Sec translocator across the inner membrane. The transportation of the passenger through the outer membrane by forming a outer memebrane pore. The virulence factors associated with T5SS passengers includes biofilm formation, adhesions, toxins, enzymes productions and cytotoxic activity (Huang and Allen. 1997).

16. Examples of T5SS effectors include: Adhesins ( B. Pertussis FHA, pertactin and Bapc , E. Coli AIDA-I and ag43 , H. Influenzae hia , HWM1, and HWM2, Shigella flexneri Icsa , Y. Enterocolitica yada) Proteases ( N. Gonorrhoeae and N. Meningitidis iga Protease, S. Flexneri sepa ) Toxins ( H. Pylori vaca ).

17. Type 6 secretion system The contact-dependent T6SS uses a phage-tail-spike-like injectisome structure to deliver effectors not only to host cells but also to competitor bacterial species, thereby giving pathogens competitive advantages within certain host growth niches. This system also includes in formation of biofilm, the quorum sensing and antibacterial toxins ( Benali et al ., 2014) .

18. T6SS effector Functions Bacteria Adherence E. coli, C. jejuni , V. parahaemolyticus Host cell invasion E. coli, C. jejuni , S. enterica, P. aeruginosa, Y. pseudotuberculosis Actin dynamics E. coli, V. cholerae Host immune responses K. pneumoniae, V .Parahaemolyticus

19. Production of plant cell wall degrading enzymes Plant cell walls consist of three major polysaccharides such as cellulose, hemicellulose and pectin, in woody and some other plants, lignin. The number of genes coding cell wall degrading enzymes varies include pectinases, proteases, cellulases and xylanases. Proteases are secreted by the T1SS, whereas the rest of the above said enzymes by the T2SS (Preston et al ., 2005) . Pectinases to be most important in pathogenesis, because they are responsible for tissue maceration by degenerating the pectic substances in the middle lamella and eventually, for cell death. Four major types of pectin degrading enzymes are secreted viz. pectate lyase, pectin lyase, pectin methyl esterase and polygalacturonase . Among these pectinase enzymes, pectate lyases (Pels) are largely involved in the virulence of soft rot Pectobacterium species.

20. Cell wall degrading enzymes are believed to play a role in pathogenesis by facilitating penetration and tissue colonization, but they are also virulence determinants responsible for development of symptom once growth of the bacteria has been started. A few Xanthomonads, e.g., X. campestris pv . campestris , the causal agent of black rot of crucifers, have genes for two pectin esterases and polygalacturonases , four pectate lyases, five xylanases and nine cellulases. Other deprived pectinolytic bacteria include A. tumefaciens , which has only four genes encoding pectinases of any form and Xylella, which has only one gene coding for a polygalacturonase .

21. Productions of bacterial toxins Toxins play a vital role in pathogenesis and parasitism of plants by several plant pathogenic bacteria. Plant pathogenic bacteria are known to produce a wide range of both specific and nonspecific host phytotoxins. Some are polypeptides, glycoproteins others are secondary metabolites. These toxins acts by using diverse mechanisms from modulating and suppressing plant defence response to alternation and inhibition of normal host cellular metabolic process. P. syringae pv . syringae , the cause of many diseases and kinds of symptoms in herbaceous and woody plants, generates necrosis-inducing phytotoxins, lipodepsipeptides, which are generally categorized into two groups, such as mycins and peptins ( Melotto et al., 2006). Chlorosis inducing phytotoxins include coronatine formed by P. syringae pv . atropurpurea , glycinea .

22. Coronatine biosynthesis plays an important role in virulence of toxin-producing P. syringae strains. Coronatine is also believed to induce hypertrophy of storage tissue, thickening of plant cell walls, accumulation of protease inhibitors, compression of thylakoids, inhibition of root elongation and stimulation of ethylene production (Alarcon- Chaidez et al., 1999).

23. Extracellular polysaccharides (EPS) may be connected to the bacterial cell as a capsule, be produced as fluidal slime, or be present in both forms. EPS play a significant role in pathogenesis of many bacteria by both direct interference with host cells and by providing resistance to oxidative stress. EPS1 is the chief virulence factor of the bacterial wilt disease caused by R . solanacearum in solanaceous crops (Milling, et al., 2011). EPS1 is a polymer made of a trimeric repeat unit consisting of N-acetyl galactosamine, deoxyl -galacturonic acid and trideoxy -d-glucose, where it is produced by the bacterium in huge quantity and constitutes more than 90% of the total polysaccharides. Xanthan, the major exopolysaccharide secreted by Xanthomonas spp., plays a key role in X. campestris pv . campestris pathogenesis. Production of extracellular polysaccharides

24. Production of phytohormones Biosynthesis of the phytohormones, auxins (e.g. indole-3-acetic acid-IAA) and cytokinins are major virulence factors for the gall-forming plant pathogenic bacteria, Pantoea agglomerans pv . Gypsophilae . Ethylene, the gaseous phytohormone formed by several microbes including plant pathogenic bacteria, can also be considered a virulence factor for some of them P. savastanoi pv . Phaseolicola ( Weingart et al., 2001).

25. Quorum sensing and biofilm production: It is a bacterial communication mechanism that regulates the density of microbial population using the gene expression in response to the environment (Kanda et al., 2011). This molecules are also known as autoinducers. Quorum-sensing signal N-acyl homoserine lactones are known to regulate numerous virulence factors including enzymes production and exopolysaccharides in many plant pathogenic bacteria. E. g. in E. amylovora a series of regulators namely MqsR , QseBC and exporter TqsA . Biofilm is a complex multilayer cellular structure attached to a tissues and embedded with an exopolysaccharide. Several plant pathogenic bacteria have been considered as biofilm producer as virulence factors including X. compestris and P. syringae (Keith et al., 2003)

26. Biofilm formation process

27. Bacterial virulence factors and the respective phytopathogens and diseases Phytopathogen Disease Virulence Factors Erwinia amylovora Fire Blight Extracellular polysaccharides (EPS) – amylovoran and levan 6-Thioguanine (6-TG) Desferrioxamine (DFO) Pseudomonas syringae pv syringae Bacterial-LeafSpot , Bacterial Speck, Bacterial Blight Coronatine (COR) Phaseolotoxin Tabtoxin Syringopeptin Syringomycin Xanthomonas spp. Cassava Bacterial Blight, Black Rot Disease, Bacterial Leaf streak Xanthan, Extracellular polysaccharides, Lipopolysaccharide, Xanthoferrin , Cyclic β-(1,2)- glucans

28. Disease symptoms caused by some bacterial pathogens of plants and representative virulence mechanisms used by these pathogens

29. Plant pathogenic bacteria employ a sophisticated array of molecular mechanisms to cause disease in plants. Key virulence factors include toxins, cell wall-degrading enzymes, and secretion systems. Secretion systems (T3SS) are crucial virulence determinants, injecting effector proteins directly into plant cells to modulate host physiology. Quorum sensing enables coordinated gene expression, facilitating virulence factor production and biofilm formation. Furthermore, some bacteria produce phytotoxins like phaseolotoxin, disrupting cellular processes and causing disease symptoms. Understanding these molecular mechanisms is crucial for developing strategies to combat plant pathogens. Targeting virulence factors, disrupting communication systems, and enhancing plant immunity are promising approaches. Future research aims to unravel complex interaction networks and develop sustainable solutions for plant disease management. CONCLUSION

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