The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.

NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Anti-CD14 mAb treatment provides therapeutic benefit after in vivo exposure to endotoxin

The presence of endotoxin from Gram-negative bacteria signals the innate immune system to up-regulate bacterial clearance and/or killing mechanisms. Paradoxically, such responses also contribute to septic shock, a clinical problem occurring with high frequency in Gram-negative septicemia. CD14 is a receptor for endotoxin (lipopolysaccharide, LPS) and is thought to have an essential role in innate immune responses to infection and thereby in the development of septic shock. Using a novel rabbit model of endotoxic shock produced by multiple exposures to endotoxin, we show that anti-rabbit CD14 mAb, which blocks LPS-CD14 binding, protects against organ injury and death even when the antibody is administered after initial exposures to LPS. In contrast, anti-rabbit tumor necrosis factor mAb treatment fails to protect when administered after LPS injections. These results support the concept that anti-CD14 treatment provides a new therapeutic window for the prevention of pathophysiologic changes that result from cumulative exposures to LPS during septic shock in man.

Establishment of an animal–bacterial association: Recruiting symbiotic vibrios from the environment

While most animal–bacterial symbioses are reestablished each successive generation, the mechanisms by which the host and its potential microbial partners ensure tissue colonization remain largely undescribed. We used the model association between the squid Euprymna scolopes and Vibrio fischeri to examine this process. This light organ symbiosis is initiated when V. fischeri cells present in the surrounding seawater enter pores on the surface of the nascent organ and colonize deep epithelia-lined crypts. We discovered that when newly hatched squid were experimentally exposed to natural seawater, the animals responded by secreting a viscous material from the pores of the organ. Animals maintained in filtered seawater produced no secretions unless Gram-negative bacteria, either living or dead, were reintroduced. The viscous material bound only lectins that are specific for either N-acetylneuraminic acid or N-acetylgalactosamine, suggesting that it was composed of a mucus-containing matrix. Complex ciliated fields on the surface of the organ produced water currents that focused the matrix into a mass that was tethered to, and suspended above, the light organ pores. When V. fischeri cells were introduced into the seawater surrounding the squid, the bacteria were drawn into its fluid-filled body cavity during ventilation and were captured in the matrix. After residing as an aggregate for several hours, the symbionts migrated into the pores and colonized the crypt epithelia. This mode of infection may be an example of a widespread strategy by which aquatic hosts increase the likelihood of successful colonization by rarely encountered symbionts.

Quorum-sensing acts at initiation of chromosomal replication in Escherichia coli

Chromosomal replication in Escherichia coli was studied by flow cytometry and was found to be inhibited by an extracellular factor present in conditioned media collected during late exponential and early stationary phase, i.e., via a quorum-sensing mechanism. Our results suggest that the inhibitory activity of the extracellular factor is exerted during initiation of DNA replication rather than during elongation. Furthermore, we present evidence that this interaction may occur directly at each of the replication forks. Unlike other quorum-sensing systems described so far for Gram-negative bacteria, this inhibitory activity does not require transcription or translation to be effective. Implications of quorum-sensing regulation of DNA replication are discussed.

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64–1.67 Mb. More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.

Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells

A number of pathogenic, Gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer bacterial membrane and the eukaryotic plasma membrane. In the pathogen Yersinia enterocolitica, the primary structure of the secreted proteins as well as of the components of the secretion machinery, both plasmid-encoded, is known. However, the mechanism of protein translocation is largely unknown. Here we show that Y. enterocolitica polymerizes a 6-kDa protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic activity. In negatively stained electron micrographs, the isolated needles were 60–80 nm long and 6–7 nm wide and contained a hollow center of about 2 nm. Our data indicate that it is the polymerization of the 6-kDa protein into these needles that provides the force to perforate the eukaryotic plasma membrane.

VanX, a bacterial d-alanyl-d-alanine dipeptidase: Resistance, immunity, or survival function?

The zinc-containing d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in d-alanyl-d-lactate (d-Ala-d-lactate) rather than d-Ala-d-Ala. The modified peptidoglycan exhibits a 1,000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to d-Ala-d-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (σs). The combined action of DdpX and the permease would permit hydrolysis of d-Ala-d-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The d-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.