Horacio Bach

Synthesis, characterization, and evaluation of antimicrobial and cytotoxic effect of silver and titanium nanoparticles

UNLABELLED Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. Nosocomial infections represent an enormous emerging problem, especially in patients with ambulatory treatment, which requires that they wear medical devices for an extended period of time. In this work, an evaluation of the antimicrobial activity of both silver and titanium nanoparticles was carried out against a panel of selected pathogenic and opportunistic microorganisms, some of them commonly associated with device-associated infections. Cytotoxicity assays monitoring DNA damage and cell viability were evaluated using human-derived monocyte cell lines. We show that silver-coated nanoparticles having a size of 20-25 nm were the most effective among all the nanoparticles assayed against the tested microorganisms. In addition, these nanoparticles showed no significant cytotoxicity, suggesting their use as antimicrobial additives in the process of fabrication of ambulatory and nonambulatory medical devices. FROM THE CLINICAL EDITOR In this study, antimicrobial activity of silver and titanium nanoparticles was evaluated against a panel of selected pathogenic and opportunistic microorganisms. Silver-coated nanoparticles of 20-25 nm size were the most effective among all the nanoparticles without significant cytotoxicity, suggesting their use as antimicrobial additives in the process of fabrication of ambulatory and nonambulatory medical devices.

Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+–ATPase to inhibit phagosome acidification

Mycobacterium tuberculosis (Mtb) pathogenicity depends on its ability to inhibit phagosome acidification and maturation processes after engulfment by macrophages. Here, we show that the secreted Mtb protein tyrosine phosphatase (PtpA) binds to subunit H of the macrophage vacuolar-H+-ATPase (V-ATPase) machinery, a multisubunit protein complex in the phagosome membrane that drives luminal acidification. Furthermore, we show that the macrophage class C vacuolar protein sorting complex, a key regulator of endosomal membrane fusion, associates with V-ATPase in phagosome maturation, suggesting a unique role for V-ATPase in coordinating phagosome–lysosome fusion. PtpA interaction with host V-ATPase is required for the previously reported dephosphorylation of VPS33B and subsequent exclusion of V-ATPase from the phagosome during Mtb infection. These findings show that inhibition of phagosome acidification in the mycobacterial phagosome is directly attributed to PtpA, a key protein needed for Mtb survival and pathogenicity within host macrophages.

Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B.

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.

Anti-biofilm activity of silver nanoparticles against different microorganisms

Biofilms confer protection from adverse environmental conditions and can be reservoirs for pathogenic organisms and sources of disease outbreaks, especially in medical devices. The goal of this research was to evaluate the anti-biofilm activities of silver nanoparticles (AgNPs) against several microorganisms of clinical interest. The antimicrobial activity of AgNPs was tested within biofilms generated under static conditions and also under high fluid shears conditions using a bioreactor. A 4-log reduction in the number of colony-forming units of Pseudomonas aeruginosa was recorded under turbulent fluid conditions in the CDC reactor on exposure to 100 mg ml−1 of AgNPs. The antibacterial activity of AgNPs on various microbial strains grown on polycarbonate membranes is reported. In conclusion, AgNPs effectively prevent the formation of biofilms and kill bacteria in established biofilms, which suggests that AgNPs could be used for prevention and treatment of biofilm-related infections. Further research and development are necessary to translate this technology into therapeutic and preventive strategies.

An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan.

ABSTRACT The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.

Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation

Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of Mr 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNγ-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNγ-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.

Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA

In Mycobacterium tuberculosis, signal transduction is mediated by 11 serine/threonine kinases, but no tyrosine kinases have been identified thus far. The protein encoded by the ORF (open reading frame) Rv2232 has been annotated as a member of the HAD (haloacid dehydrogenase-like hydrolase) superfamily, which includes phosphatases, phosphomanno- and phosphogluco-mutases, and haloacid dehydrogenases. In the present paper, we report, on the basis of biochemical and mutational analyses, that the Rv2232-encoded protein, named protein tyrosine kinase A (PtkA) is a bona fide protein tyrosine kinase. The cognate substrate of PtkA is the secreted protein tyrosine phosphatase A (PtpA).

Fusidic acid and rifampicin co-loaded PLGA nanofibers for the prevention of orthopedic implant associated infections.

Implant-associated infections following invasive orthopedic surgery are a major clinical problem, and are one of the primary causes of joint failure following total joint arthroplasty. Current strategies using perioperative antibiotics have been met with little clinical success and have resulted in various systemic toxicities and the promotion of antibiotic resistant microorganisms. Here we report the development of a biodegradable localized delivery system using poly(D,L-lactic acid-co-glycolic acid) (PLGA) for the combinatorial release of fusidic acid (FA) (or its sodium salt; SF) and rifampicin (RIF) using electrospinning. The drug-loaded formulations showed good antibiotic encapsulation (~75%-100%), and a biphasic drug release profile. All dual-loaded formulations showed direct antimicrobial activity in vitro against Staphylococcus epidermidis, and two strains of methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, lead formulations containing 10% (w/w) FA/SF and 5% (w/w) RIF were able to prevent the adherence of MRSA to a titanium implant in an in vivo rodent model of subcutaneous implant-associated infection.