Investigation of the role of type IV Aeromonas pilus (Tap) in the pathogenesis of Aeromonas gastrointestinal infection.

Although there is substantial evidence that type IV pili purified from diarrhea-associated Aeromonas species (designated Bfp for bundle-forming pilus) are intestinal colonization factors (S. M. Kirov, L. A. O'Donovan, and K. Sanderson, Infect. Immun. 67:5447-5454, 1999), nothing is known regarding the function of a second family of Aeromonas type IV pili (designated Tap for type IV Aeromonas pilus), identified following the cloning of a pilus biogenesis gene cluster tapABCD. Related pilus gene clusters are widely conserved among gram-negative bacteria, but their significance for virulence has been controversial. To investigate the role of Tap pili in Aeromonas pathogenesis, mutants of Aeromonas strains (a fish isolate of A. hydrophila and a human dysenteric isolate of A. veronii bv. sobria) were prepared by insertional inactivation of the tapA gene which encodes the type IV pilus subunit protein, TapA. Exotoxic activities were unaffected by the mutation in tapA. Inactivation of tapA had no effect on the bacterial adherence of these two isolates to HEp-2 cells. For the A. veronii bv. sobria isolate, adhesion to Henle 407 intestinal cells and to human intestinal tissue was also unaffected. There was no significant effect on the duration of colonization or incidence of diarrhea when the A. veronii bv. sobria strain was tested in the removable intestinal tie adult rabbit diarrhea model or on its ability to colonize infant mice. Evidence was obtained that demonstrated that TapA was expressed by both Aeromonas species and was present on the cell surface, although if assembled into pili this pilus type appears to be an uncommon one under standard bacterial growth conditions. Further studies into factors which may influence Tap expression are required, but the present study suggests that Tap pili may not be as significant as Bfp pili for Aeromonas intestinal colonization.

The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice.

Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

Regulation of the "tetCD" genes of transposon Tn10.

In addition to the genes involved in tetracycline resistance, the loop region of the composite transposon Tn10 contains two other known genes, tetC and tetD, whose functions are unclear. Using primarily a genetic approach, we examined tetCD gene expression and regulation. The tetC gene product, TetC, is a diffusible repressor of both tetC and tetD transcription. Despite an earlier claim by others, we do not detect induction of either tetC or tetD by tetracycline (Tc) or several of its analogs. Although the 5' ends of the tetC and tetD messages overlap due to transcription from convergent promoters, we find no evidence for anti-sense RNA control. The operator for the TetC repressor has been localized. We also demonstrate that transcription from the tetD promoter probably terminates within IS10-Right and does not apparently interfere with Tn10 or IS10-Right transposition or its regulation.

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.

Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack.

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.

[Furunculoid myiasis caused by Dermatobia hominis]. / Miásis forunculoide por "dermatobia hominis"

The authors report a case of forunculoid miasis by "Dermatobia hominis" ("Ura" fly). a process that being not very common in the Argentine Republic is sometimes misjudged as a furunculosis. The patient reported, a white woman, 51, travelled to a zone where this parasitose is endemid. After some time of her stay there, she noted three small nodes in her vulvar region. They grew on until reaching nearly the size of a walnut. At her first consultation with a gynecologist it was taken as a case of furuncolosis and treated with antibiotics. As the treatment didn't prove succesful, she asked for dermatological advice. It was then possible to verify the presence of three nodes. In the central hole of one of them, there could be seen a whitish body that soon disappeared inwards. This lead to the diagnosis of forunculoid miasis. An accurate and easy-to-perform treatment was carried out: a 1% solution of formaldehyde in distilled water was infused through the central hole of each node. This determined the larva's caudal extremity to appear in search of air. It was taken advantage of this situation to remove the larvae with a pair of nippers. "Dermatobia hominis" is widely spread in the warm-climated Argentine northern provinces of Chaco, Formosa and Misiones, as well as in Brazil, Paraguay and some regions of Uruguay. It is also known under the name of "Ura". It is a large fly, yellow faced, with dark-blue thorax; an almost rombic metallic-blue abdomen, orange antennae and legs, and dark-brown wings. It lives on the nutrients spared during its larval stage and both fecundation and oviposition, take place along its week-long life. The fecundated female catches different haematophagus insects on the wing, sticking sets of eggs to their abdomens. An embryo developes in each egg, being ready to emerge in a week but does this only when the bearer insect settles on a homeothermal being piercing and penetrating the skin of the new guest in about 10 minutes.