Madalena Pimentel

Antimicrobial activity of Guinea-Bissau traditional remedies

The ethanolic extracts of twelve plants selected through ethnomedical survey in Guinea-Bissau were investigated for their in vitro antimicrobial properties over ten bacteria and Candida albicans, using agar diffusion and dilution methods. All the tested extracts showed some activity against at least one of the bacteria. Most of the extracts (79%) showed activity against Staphylococcus aureus and only one (Cryptolepis sanguinolenta) against Escherichia coli. Cryptolepis sanguinolenta and Terminalia macroptera root extracts showed some activity against Candida albicans as well as showing an interesting profile of activity against most of the enteropathogen microorganisms. Inhibition zones against Staphylococcus aureus were localised on extract chromatograms by bioautographic techniques.

Antimicrobial activity of Terminalia macroptera root

Terminalia macroptera Guill et Perr. (Combretaceae) is a medicinal plant used in Guinea-Bissau and other West African countries to treat infectious diseases. The ethanol extract from T. macroptera decorticated root and their liquid-liquid partition fractions, were screened for antimicrobial activity, by the twofold serial microdilution assay against seven reference bacterial strains and against Candida albicans. The extract and fractions showed some activity against at least one of the test microorganisms. The best results were obtained against Shigella dysenteriae and Vibrio cholerae. The minimum inhibitory concentrations (MIC) of T. macroptera ethanol extract were also determined for about 100 clinical strains of Campylobacter sp., Escherichia coli, Salmonella sp., Shigella sp. and Vibrio cholerae. The ethanol extract activity against Campylobacter strains is similar to co-trimoxazole, higher than sulfamethoxazole but lower than tetracycline, erythromycin, ampicillin and streptomycin. Ellagitannins are the major compounds in the extract and active fractions. The obtained results suggest a potential importance of this medicinal plant in the treatment of enteric diseases, particularly in Campylobacter infections.

Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

A mycobacteriophage Ms6 strong promoter region (P(lys)) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem sigma(70)-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P(lys) drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that beta-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity.

dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan.

Like most double‐stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin‐endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone‐like protein, in a holin‐independent manner.

Cryptolepis sanguinolenta activity against diarrhoeal bacteria

Cryptolepine is the main alkaloid of Cryptolepis sanguinolenta (Lindl.) Schlechter, a plant used in traditional medicine in West Africa. The minimal inhibitory concentrations (MICs) of cryptolepine, ethanol and aqueous extracts of Cryptolepis sanguinolenta root were determined for 65 strains of Campylobacter jejuni, 41 strains of Campylobacter coli isolated from sporadic cases of gastroenteritis in Portugal and 86 strains of Vibrio cholerae isolated from patients with enteric infections in Angola, Brazil and Portugal. The ethanol extract activity against Campylobacter strains (MIC90% = 25 micrograms/ml) is higher than that of co-trimoxazole and sulfamethoxazole and Campylobacter strains susceptibility for cryptolepine (MIC90% = 12.5 micrograms/ml) is equal for ampicillin. The ethanol extract and cryptolepine show some activity against the Vibrio cholerae strains, although their activities are lower than that of tetracycline. The results suggest that these roots could be a therapeutic alternative for bacterial etiologic diarrhoea in West Africa.

A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6.

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA 241) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin384) or the shorter (Lysin241) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various Gram-positive bacteria and mycobacteria.

Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene (gp5) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli. One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5, although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6(Δgp4)) caused slightly accelerated lysis, whereas an Ms6(Δgp5) deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.