Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica.

Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA' (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

Cyclic nucleotide signaling in Mycobacterium tuberculosis: an expanding repertoire.

Mycobacterium tuberculosis (Mtb) is one of the most successful microbial pathogens, and currently infects over a quarter of the world's population. Mtb's success depends on the ability of the bacterium to sense and respond to dynamic and hostile environments within the host, including the ability to regulate bacterial metabolism and interactions with the host immune system. One of the ways Mtb senses and responds to conditions it faces during infection is through the concerted action of multiple cyclic nucleotide signaling pathways. This review will describe how Mtb uses cyclic AMP, cyclic di-AMP and cyclic di-GMP to regulate important physiological processes, and how these signaling pathways can be exploited for the development of novel thereapeutics and vaccines.

Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling.

Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

Relationship of Soil Properties and Sugarcane Yields to Red Stripe in Louisiana.

Symptoms of red stripe disease caused by Acidovorax avenae subsp. avenae in Louisiana between 1985 and 2010 were limited to the leaf stripe form, which caused no apparent yield loss. During 2010, the more severe top rot form was observed, and a study was initiated to investigate the distribution of red stripe in the field and determine its effects on cane and sugar yields. Soil properties data, red stripe incidence, and sugarcane yields were all highly variable and were not randomly distributed in the field. Combined harvest data showed a negative correlation between yield components and red stripe incidence, with the strongest relationship between sucrose per metric ton and disease incidence. Red stripe incidence was positively correlated with several soil properties, including phosphorus, potassium, zinc, and calcium. Red stripe incidence also was found to increase with increasing nitrogen rate, with the greatest effects in heavy soils. Results also indicated that using red-stripe-infected cane as a seed source can significantly decrease shoot emergence, stalk population, and subsequent cane and sugar yields. These combined data suggest that red stripe disease can exhibit a highly variable rate of infection in commercial sugarcane fields and may also significantly decrease sugar yields.

Detecting Sugarcane yellow leaf virus infection in asymptomatic leaves with hyperspectral remote sensing and associated leaf pigment changes.

Sugarcane infected with Sugarcane yellow leaf virus (SCYLV) rarely produces visual symptoms until late in the growing season. High-resolution, hyperspectral reflectance data from SCYLV-infected and non-infected leaves of two cultivars, LCP 85-384 and Ho 95-988, were measured and analyzed on 13 July, 12 October, and 4 November 2005. All plants were asymptomatic. Infection was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Results from discriminant analysis showed that leaf reflectance was effective at predicting SCYLV infection in 73% of the cases in both cultivars using resubstitution and 63% and 62% in LCP 85-384 and Ho 95-988, respectively, using cross-validation. Predictive equations were improved when data from sampling dates were analyzed individually. SCYLV infection influenced the concentration of several leaf pigments including violaxanthin, beta-carotene, neoxanthin, and chlorophyll a. Pigment data were effective at predicting SCYLV infection in 80% of the samples in the combined data set using the derived discriminant function with resubstitution, and 71% with cross-validation. Although further research is needed to improve the accuracy of the predictive equations, the results of this study demonstrate the potential application of hyperspectral remote sensing as a rapid, field-based method of identifying SCYLV-infected sugarcane plants prior to symptom expression.

Relationship between sugarcane rust severity and soil properties in louisiana.

ABSTRACT The extent of spatial and temporal variability of sugarcane rust (Puccinia melanocephala) infestation was related to variation in soil properties in five commercial fields of sugarcane (interspecific hybrids of Saccharum spp., cv. LCP 85-384) in southern Louisiana. Sugarcane fields were grid-soil sampled at several intensities and rust ratings were collected at each point over 6 to 7 weeks. Soil properties exhibited significant variability (coefficients of variation = 9 to 70.1%) and were spatially correlated in 39 of 40 cases with a range of spatial correlation varying from 39 to 201 m. Rust ratings were spatially correlated in 32 of 33 cases, with a range varying from 29 to 241 m. Rust ratings were correlated with several soil properties, most notably soil phosphorus (r = 0.40 to 0.81) and soil sulfur (r = 0.36 to 0.68). Multiple linear regression analysis resulted in coefficients of determination that ranged from 0.22 to 0.73, and discriminant analysis further improved the overall predictive ability of rust models. Finally, contour plots of soil properties and rust levels clearly suggested a link between these two parameters. These combined data suggest that sugarcane growers that apply fertilizer in excess of plant requirements will increase the incidence and severity of rust infestations in their fields.