Mechanism of lipid bilayer perturbation by bactericidal membrane-active small molecules.

Membrane-active small molecules (MASMs) are small organic molecules designed to reproduce the fundamental physicochemical properties of natural antimicrobial peptides: their cationic charge and amphiphilic character. This class of compounds has a promising broad range of antimicrobial activity and, at the same time, solves some major limitations of the peptides, such as their high production costs and low in vivo stability. Most cationic antimicrobial peptides act by accumulating on the surface of bacterial membranes and causing the formation of defects when a threshold is reached. Due to the drastically different structures of the two classes of molecules, it is not obvious that small-molecule antimicrobials act in the same way as natural peptides, and very few data are available on this aspect. Here we combined spectroscopic studies and molecular dynamics simulations to characterize the mechanism of action of two different MASMs. Our results show that, notwithstanding their simple structure, these molecules act just like antimicrobial peptides. They bind to the membrane surface, below the head-groups, and insert their apolar moieties in the core of the bilayer. Like many natural peptides, they cause the formation of defects when they reach a high coverage of the membrane surface. In addition, they cause membrane aggregation, and this property could contribute to their antimicrobial activity.

Isosteric substitution in cationic-amphiphilic polymers reveals an important role for hydrogen bonding in bacterial membrane interactions.

Biomimetic antibacterial polymers, the functional mimics of antimicrobial peptides (AMPs), targeting the bacterial cell membrane have been developed to combat the problem of antibiotic resistance. Amphiphilicity, a balance of cationic charge and hydrophobicity, in these polymers has been shown to be pivotal for their selective interactions with anionic lipid membranes of bacteria instead of zwitterionic mammalian (human erythrocyte) membranes. However, it is unclear if and to what extent hydrogen bonding in amphiphilic antibacterial polymers contributes to this membrane binding specificity. To address this, we employ isosteric substitution of ester with amide moieties that differ in their potency for hydrogen bonding in the side chains of N-alkyl maleimide based amphiphilic polymers. Our studies reveal that amide polymer (AC3P) is a potent antibacterial agent with high membrane-disrupting properties compared to its ester counterpart (EC3P). To understand these differences we performed bio-physical experiments and molecular dynamics (MD) simulations which showed strong interactions of AC3P including hydrogen bonding with lipid head groups of bacterial model lipid bilayers, that are absent in EC3P, make them selective for bacterial membranes. Mechanistic investigations of these polymers in bacteria revealed specific membrane disruptive activity leading to the delocalization of cell division related proteins. This unprecedented and unique concept provides an understanding of bacterial membrane interactions highlighting the role of hydrogen bonding. Thus, these findings will have significant implications in efficient design of potent membrane-active agents.

Monoarticular corticosteroid injection versus systemic administration in the treatment of rheumatoid arthritis patients: a randomized double-blind controlled study.

OBJECTIVE: To compare the efficacy and safety of intraarticular glucocorticoid injection to its systemic use for treatment of knee synovitis in rheumatoid patients. METHODS: A randomized double-blind controlled study was conducted including 60 patients with RA. Patients were randomized to receive either a single intraarticular knee injection with triamcinolone hexacetonide 60 mg (3 ml) and xylocaine chloride 2% (1 ml) associated to a single intramuscular injection of 1 ml of xylocaine chloride 2% (IAI group) or 1 ml of xylocaine chloride 2% by intraarticular injection and a intramuscular injection of triamcinolone acetonide 60 mg (3 ml) and xylocaine chloride 2% (1 ml) (IM group). All patients were blindfolded for the procedure. Evaluations were performed at baseline and 1, 4, 8 and 12 weeks post-intervention. The following instruments were used: VAS for knee pain, as primary outcome, VAS for knee morning stiffness and edema; the ACR 20, 50 and 70% improvement criteria; knee circumference and goniometry; Likert's scale of improvement; daily use of oral glucocorticoid and NSAIDs, blood pressure and adverse effects. RESULTS: Patients in the IAI group had significantly better results for VAS for knee pain, edema and morning stiffness as well as for improvement evaluation after intervention according to the patient (p<0.001) and physician (p=0.02). CONCLUSION: Our results demonstrate that intraarticular injection with glucocorticoids is superior to its systemic use for the management of monoarticular synovitis in rheumatoid patients. The intraarticular approach showed better results in terms of local inflammatory variables and improvement evaluation by the patient and physician.

Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly.

Progenies from some wild-caught females of Drosophila willistoni and three other sibling species are entirely female. The proclivity for production of unisexual female progeny by these flies was named the sex ratio (SR) trait and was originally thought to be genetic. However, experiments in the laboratory of Donald F. Poulson in the early 1960s demonstrated that this 'trait' was vertically transmitted and infectious, in that it could be artificially transferred by injection from infected females to non-infected females. Motile, helical micro-organisms were observed in females showing the trait. In 1979, the SR organisms were designated as group II in the informal spiroplasma classification system. The organisms proved to be extremely fastidious, but were eventually cultivated in a very complex cell-free medium (H-2) after initial co-cultivation with insect cells. Cultivation in the H-2 medium and the subsequent availability of a triply cloned strain (DW-1T) permitted comparative studies. Cells of strain DW-1T were helical, motile filaments 200-250 nm in diameter and were bound by a single trilaminar membrane. Cells plated on 1.8% Noble agar formed small satellite-free colonies 60-70 microns in diameter with dense centres and uneven edges. The temperature range for growth was 26-30 degrees C; optimum growth occurred at 30 degrees C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 100 nm, pores. Reciprocal serological comparisons of strain DW-1T with representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-1T was used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DW-1T and representatives of group I sub-groups. The genome size of strain DW-1T was 2040 kbp, as determined by PFGE. The G + C content was 26 +/- 1 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-1T is separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasmas to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-1T (ATCC 43153T), are designated Spiroplasma poulsonii.

Revised group classification of the genus Spiroplasma.

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

Spiroplasma platyhelix sp. nov., a new mollicute with unusual morphology and genome size from the dragonfly Pachydiplax longipennis.

Spiroplasma strain PALS-1T from the gut of the dragonfly Pachydiplax longipennis was shown to be distinct from other species, groups, and subgroups of the genus Spiroplasma as determined by reciprocal serological metabolism inhibition and deformation tests. However, this strain cross-reacted extensively with representatives of other groups when it was used as an antigen. Electron microscopy of cells of strain PALS-1T revealed cells surrounded by a single cytoplasmic membrane. Light microscopy revealed helical cells that exhibited twisting motility rather than rotatory or flexing motility. Variations in the tightness of coiling were transmitted from one end of the helix to the other. The strain was resistant to penicillin, which confirmed that no cell wall was present. The organism grew well in M1D and SP-4 liquid media under either aerobic or anaerobic conditions. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. The optimum temperature for growth was 30 degrees C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32 degrees C. Strain PALS-1T catabolized glucose and hydrolyzed arginine but not urea. The guanine-plus-cytosine content of the DNA was 29 +/- 1 mol%. The genome size was 780 kbp, the smallest genome size in the genus Spiroplasma. Strain PALS-1 (= ATCC 51748) is designated the type strain of a new species, Spiroplasma platyhelix.

Spiroplasma diabroticae sp. nov., from the southern corn rootworm beetle, Diabrotica undecimpunctata (Coleoptera:Chrysomelidae).

Spiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32 degrees C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41 degrees C but failed to grow at 5 or 43 degrees C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 +/- 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.

Spiroplasma corruscae sp. nov., from a firefly beetle (Coleoptera: Lampyridae) and tabanid flies (Diptera: Tabanidae).

Spiroplasma strain EC-1T (T = type strain), which was isolated from the gut of a lampyrid beetle (Ellychnia corrusca) in Maryland, was serologically distinct from other spiroplasma species and groups. Similar strains were obtained from other E. corrusca specimens, and, later, numerous isolates of similar or partially related strains were obtained from several species of tabanid files. Cells of strain EC-1T were helical, motile filaments that were bound by a single cytoplasmic membrane, and there was no evidence of a cell wall. The cells were filterable through 220-nm-pore-size membrane filters but not through 100-nm-pore-size membrane filters. The organism was absolutely resistant to penicillin (1,000 U/ml) and required sterol for growth. Strain EC-1T grew well in M1D and SP-4 liquid media and could be cultivated in the Edward formulation of conventional mycoplasma medium and in 1% serum fraction medium. Optimal growth occurred at 32 degrees C (doubling time, 1.5 h). Strain EC-1T multiplied at 10 to 41 degrees C, but not at 5 or 43 degrees C. This organism produced acid from glucose, but did not hydrolyze arginine or utilize urea. The guanine-plus-cytosine content of the DNA was determined to be 26.3 mol% by the melting temperature method and 27.0 mol% by the buoyant density method. As a result of our studies, strain EC-1 (= ATCC 43212) is designated the type strain of a new species, Spiroplasma corruscae.

Improved cultivation systems for isolation of the colorado potato beetle spiroplasma.

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.

Spiroplasma syrphidicola sp. nov., from a syrphid fly (Diptera: Syrphidae).

Spiroplasma sp. strain EA-1(T) (T = type strain) (subgroup VIII-1), which was isolated from the syrphid fly Eristalis arbustorum, was serologically distinct from other spiroplasma species, groups, and subgroups, The cells of this strain, as revealed by dark-field light microscopy, were short, helical, and motile. An electron microscopic examination revealed wall-less cells delimited by a single membrane. The unusually short cells passed through 220-nm filter pores with no reduction in titer. The organisms grew well in SM-1, M1D, and SP-4 liquid media. Growth also occurred in conventional horse serum medium and 1% serum fraction medium. Strain EA-1(T) grew at temperatures between 10 and 41 degrees C, and optimum growth occurred at 32 degrees C. The doubling time at the optimal temperature was 1.0 h. The strain catabolized glucose and hydrolyzed arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 30 +/- 1 mol%. The genome size was about 1,230 kbp. Strain Ea-1 (= ATCC 33826), which represents subgroup VIII-1, is designated the type strain of a new species, Spiroplasma syrphidicola.

Temperature Ranges, Growth Optima, and Growth Rates of Spiroplasma (Spiroplasmataceae, class Mollicutes) Species

A new method was developed for determination of the doubling times of spiroplasmas. In this procedure, the time required for medium acidification of tubes in tenfold dilution series was recorded. Sixty-four spiroplasma strains, representing 24 groups and 11 subgroups, were studied. Eight strains representing putative new groups were also included in the study. Doubling times at 5, 10, 15, 20, 25, 30, 32, 37, 41, and 43°C were determined. The range of temperatures for spiroplasma growth was 5°-41°C. Twenty-three spiroplasmas had optima of 30°C, 29 had optima of 32°C, and 13 had optima of 37°C. The fastest growing spiroplasma was the MQ-4 strain (group XI), with a doubling time at optimal temperature of 0.6 h. The slowest was the Jamaican corn stunt strain B655 (subgroup I-3), with an optimal doubling time of 36.7 h. Spiroplasma strain B31 (group IV) had the widest range (5°-41°C), while the DW-1 strain and some subgroup I-3 strains had the narrowest, growing only at 25° and 30°C. Some spiroplasmas grew well at 41°C, but none grew at 43°C. The ability of spiroplasmas to withstand a wide range of temperatures may reflect the conditions to which they are exposed in nature, including the temperatures of the insect, tick, and/or plant hosts in which they are carried and the plant surfaces from which they may be acquired by arthropods.

Spiroplasma velocicrescens sp. nov., from the vespid wasp Monobia quadridens.

Spiroplasma strain MQ-4T (T = type strain), which was isolated from the hemolymph of the vespid wasp Monobia quadridens, was serologically distinct from other spiroplasma species, groups, putative groups, and subgroups. Each strain MQ-4T cell was helical and motile and was surrounded by a single cytoplasmic membrane; there was no evidence of a cell wall. The strain grew well in 1% serum fraction medium, as well as in SM-1, M1D, and SP-4 liquid media, under both aerobic and anaerobic conditions. Strain MQ-4T grew at temperatures ranging from 10 to 41 degrees C but did not grow at 43 degrees C. The strain grew optimally at 37 degrees C with a doubling time of 0.6 h, the shortest doubling time recorded for any spiroplasma. Strain MQ-4T catabolized glucose and arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was about 27.5 +/- 1 mol%. The genome size was 1,480 kbp (940 MDa). Strain MQ-4 (= ATCC 35262) is designated the type strain of a new species, Spiroplasma velocicrescens.