Decrease of ESKAPE virulence with a cationic heme-mimetic gallium porphyrin photosensitizer: The Trojan horse strategy that could help address antimicrobial resistance.

INTRODUCTION: Emerging antibiotic resistance among bacterial pathogens has forced an urgent need for alternative non-antibiotic strategies development that could combat drug resistant-associated infections. Suppression of virulence of ESKAPE pathogens' by targeting multiple virulence traits provides a promising approach. OBJECTIVES: Here we propose an iron-blocking antibacterial therapy based on a cationic heme-mimetic gallium porphyrin (GaCHP), which antibacterial efficacy could be further enhanced by photodynamic inactivation. METHODS: We used gallium heme mimetic porphyrin (GaCHP) excited with light to significantly reduce microbial viability and suppress both the expression and biological activity of several virulence traits of both Gram-positive and Gram-negative ESKAPE representatives, i.e., S. aureus and P. aeruginosa. Moreover, further improvement of the proposed strategy by combining it with routinely used antimicrobials to resensitize the microbes to antibiotics and provide enhanced bactericidal efficacy was investigated. RESULTS: The proposed strategy led to substantial inactivation of critical priority pathogens and has been evidenced to suppress the expression and biological activity of multiple virulence factors in S. aureus and P. aeruginosa. Finally, the combination of GaCHP phototreatment and antibiotics resulted in promising strategy to overcome antibiotic resistance of the studied microbes and to enhance disinfection of drug resistant pathogens. CONCLUSION: Lastly, considering high safety aspects of the proposed treatment toward host cells, i.e., lack of mutagenicity, no dark toxicity and mild phototoxicity, we describe an efficient alternative that simultaneously suppresses the functionality of multiple virulence factors in ESKAPE pathogens.

Development of Antimicrobial Phototreatment Tolerance: Why the Methodology Matters.

Due to rapidly growing antimicrobial resistance, there is an urgent need to develop alternative, non-antibiotic strategies. Recently, numerous light-based approaches, demonstrating killing efficacy regardless of microbial drug resistance, have gained wide attention and are considered some of the most promising antimicrobial modalities. These light-based therapies include five treatments for which high bactericidal activity was demonstrated using numerous in vitro and in vivo studies: antimicrobial blue light (aBL), antimicrobial photodynamic inactivation (aPDI), pulsed light (PL), cold atmospheric plasma (CAP), and ultraviolet (UV) light. Based on their multitarget activity leading to deleterious effects to numerous cell structures-i.e., cell envelopes, proteins, lipids, and genetic material-light-based treatments are considered to have a low risk for the development of tolerance and/or resistance. Nevertheless, the most recent studies indicate that repetitive sublethal phototreatment may provoke tolerance development, but there is no standard methodology for the proper evaluation of this phenomenon. The statement concerning the lack of development of resistance to these modalities seem to be justified; however, the most significant motivation for this review paper was to critically discuss existing dogma concerning the lack of tolerance development, indicating that its assessment is more complex and requires better terminology and methodology.

Photoinactivation of ESKAPE pathogens: overview of novel therapeutic strategy.

The emergence of antimicrobial drug resistance requires development of alternative therapeutic options. Multidrug-resistant strains of Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter spp. are still the most commonly identified antimicrobial-resistant pathogens. These microorganisms are part of the so-called 'ESKAPE' pathogens to emphasize that they currently cause the majority of hospital acquired infections and effectively 'escape' the effects of antibacterial drugs. Thus, alternative, safer and more efficient antimicrobial strategies are urgently needed, especially against 'ESKAPE' superbugs. Antimicrobial photodynamic inactivation is a therapeutic option used in the treatment of infectious diseases. It is based on a combination of a photosensitizer, light and oxygen to remove highly metabolically active cells.

Photoinactivation of Staphylococcus aureus using protoporphyrin IX: the role of haem-regulated transporter HrtA.

Light- and photosensitiser-based antimicrobial photodynamic therapy is a very promising approach to the control of microbial infections. How the phenotypic features of a microorganism affect its response to photosensitiser-based photokilling represents an area of substantial research interest. To understand the mechanisms governing the phenomenon of a strain-dependent response to photodynamic inactivation (PDI), we analysed the possible role of the membrane-located haem transporter HrtA in Staphylococcus aureus. We used a S. aureus strains with an inactivated component of the haem-regulated transporter, HrtA, along with its wild-type counterpart to determine differences in PDI outcome and photosensitiser uptake between the studied isogenic strains. We observed that a lack of HrtA protein potentiates the phototoxic effect towards S. aureus but only when extracellular protoporphyrin IX is used. The observed effect may depend on the function of the HrtA transporter but is likely to result from changed membrane properties following the absence of the protein in the membrane. This indicates that disturbing the membrane properties is an attractive method for improving the efficacy of the photodynamic inactivation of microorganisms.

Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies.

A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections.

The agr function and polymorphism: impact on Staphylococcus aureus susceptibility to photoinactivation.

Staphylococcus aureus is an important human pathogen that causes healthcare-associated and community-acquired infections. Moreover, the growing prevalence of multiresistant strains requires the development of alternative methods to antibiotic therapy. One effective therapeutic option may be antimicrobial photodynamic inactivation (aPDI). Recently, S. aureus strain-dependent response to PDI was demonstrated, although the mechanism underlying this phenomenon remains unexplained. The aim of the current study was to investigate statistically relevant correlations between the functionality and polymorphisms of agr gene determined for 750 methicillin-susceptible and methicillin-resistant S. aureus strains and their responses to photodynamic inactivation using protoporphyrin IX. An AluI and RsaI digestion of the agr gene PCR product revealed existing correlations between the determined digestion profiles (designations used for the first time) and the PDI response. Moreover, the functionality of the agr system affected S. aureus susceptibility to PDI. Based on our results, we conclude that the agr gene may be a genetic factor affecting the strain dependent response to PDI.

Discovering the mechanisms of strain-dependent response of Staphylococcus aureus to photoinactivation: oxidative stress toleration, endogenous porphyrin level and strain's virulence.

BACKGROUND: Staphylococcus aureus is generally known to be susceptible to photoinactivation. However, the phenomenon of its strain-dependent response to photodynamic treatment has been reported. Moreover, the factors determining the emerging variation among strains according to photoinactivation remain unclear. METHODS: This work aimed to investigate any relevant correlation between bacterial toleration of oxidative stress, porphyrin level, photosensitizer uptake and strain's virulence of studied methicillin-susceptible and methicillin-resistant S. aureus strains and their response to photodynamic inactivation (using protoporphyrin diarginate, toluidine blue O and 5-aminolevulinic acid). RESULTS: Obtained data let to demonstrate that studied factors have limited impact on strain response to PDI. However, we have shown that multicomponent sensitizing agent i.e. consisted of PPArg2, ALA and TBO would eliminate the S. aureus elevated resistance to photoinactivation and that both highly virulent and low virulent S. aureus strains could be easily eradicated with the use of PDI. Moreover, we have shown that photodynamic inactivation could decrease the virulence of S. aureus extracellular fraction. CONCLUSION: The mechanism underlying strain-dependent response to photoinactivation is complex and multifactorial nevertheless with the use of several sensitizing agents the elevated resistance to photodynamic treatment can be omitted.

Host response to the presence of Helicobacter spp. DNA in the liver of patients with chronic liver diseases.

Literature data indicate an association between the presence of Helicobacter spp. in the liver and the development of hepatocellular carcinoma (HCC). However, the role of H. pylori infections in chronic liver diseases (CLD) remains controversial. The aim of this study was to detect Helicobacter spp. DNA in patients with CLD, and to investigate the host response to the presence of the bacterium in the liver. Helicobacter spp. DNA was detected in 59% samples. H.pylori was the most prevalent species (94%). We estimated the expression level of IL-1 and IL-8 genes. The presence of Helicobacter spp. did not have a significant effect on the gene expression of IL-8 and IL-1.

The connection between agr and SCCmec elements of Staphylococcus aureus strains and their response to photodynamic inactivation.

OBJECTIVE: The aim of this work was to analyze the presence of specific types of agr and SCCmec in Staphylococcus aureus strains and to determine the correlation between these types of genes and the response of S. aureus strains to photodynamic inactivation. BACKGROUND: S. aureus is an important human pathogen that is still one of the most common etiological factors of nosocomial infections. The genetic factor connected with high pathogenicity of S. aureus strains is the agr locus, which encodes a molecule responsible for activation of virulence genes. The characteristic feature of strains resistant to methicillin (MRSA) is the presence of the gene determining the resistance to ß-lactam antibiotics. This gene is a part of a mobile genetic element known as Staphylococcal Chromosome Cassette mec (SCCmec). Polymorphic differences in the agr locus and SCCmec cassette enable classification of strains into different groups. MATERIALS AND METHODS: We cultured and incubated each strain with defined dose of photosensitizer (protoporphyrin diarginate). Next, strains were irradiated with a red light at a dose of 12 J/cm(2). After an 18-h incubation, the Colony Forming Units were counted and the results were analyzed statistically. Furthermore, the genetic profile of the studied strains was determined with the use of the Multiplex PCR reaction both for agr and SCCmec elements. RESULTS: The results agreed with previous data, confirming that the response to photodynamic inactivation varies among different S. aureus strains. We also found a connection between some of the agr and SCCmec groups and the response of analyzed S. aureus strains to photoinactivation. CONCLUSION: Unfortunately, those relations are not specific enough to determine a diagnostically important pattern, which could enable predictions of strain response to PDI. Nevertheless, we can conclude that the connection between the response of S. aureus strains to photoinactivation and the strain specific agr/SCCmec pattern could be observed.

[Helicobacter spp. infections in chronic liver damage]. / Zakazenia bakteriami rodzaju Helicobacter spp. w przewleklym uszkodzeniu watroby.

Liver is a key organ responsible for organism's homeostasis. A proper function of this organ is crucial for detoxification of metabolic products and regulation of metabolic processes of macromolecules (proteins, lipids, carbohydrates). The most important infectious factors, leading to liver damage, are primary hepatotropic viruses, particularly those causing chronic inflammation of the organ (HBV, HCV, HDV), which may subsequently cause cirrhosis and/or primary hepatocellular carcinoma. There has been a growing interest in Helicobacter spp. liver infections as a potential factor promoting injury of the organ towards hepatocellular carcinoma. The association between hepatocellular carcinoma and the presence of Helicobacters in the liver has been well documented in animal models (Helicobacter hepaticus versus liver cancer in mice). Some reports also indicate similar association in humans, where the presence of Helicobacter antigens in patients with liver cancer is detected more often in comparison to healthy or chronically infected population. Although the molecular mechanisms underlying such a phenomenon are not well known, the knowledge on this subject has considerably increased during recent years. The review presents data on the association between the presence of Helicobacter spp. in the liver and injuries of the organ, as well as the role that is played by the bacteria in chronic liver diseases.

Detection of Helicobacter rodentium-like DNA in the liver tissue of patients with chronic liver diseases by polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequence analysis.

Many Helicobacter spp. were isolated from the stomach, intestinal tract, and liver of different animals and humans. The association between Helicobacter spp. and hepatobiliary diseases, including hepatocellular carcinoma, was thoroughly examined, indicating a potential role of the bacteria in the progression toward cancer. In our work, we screened 97 liver biopsies from patients with chronic liver diseases for the presence of Helicobacter spp. DNA. With the use of genus-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing, we found that the majority of Helicobacter spp. DNA detected was similar to Helicobacter rodentium DNA (71%). The DNA of other detected Helicobacter spp. was similar to Helicobacter pylori DNA. This is the first indication of H. rodentium-like DNA presence in human liver tissue. We also conclude that PCR-DGGE is a useful screening method for assigning species designation and heterogeneity.

Functional analysis of MmeI from methanol utilizer Methylophilus methylotrophus, a subtype IIC restriction-modification enzyme related to type I enzymes.

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

Binding of MmeI restriction-modification enzyme to its specific recognition sequence is stimulated by S-adenosyl-L-methionine.

Restriction endonucleases serve as a very good model for studying specific protein-DNA interaction. MmeI is a very interesting restriction endonuclease, but although it is useful in Serial Analysis of Gene Expression, still very little is known about the mechanism of its interaction with DNA. MmeI is a unique enzyme as besides cleaving DNA it also methylates specific sequence. For endonucleolytic activity MmeI requires Mg(II) and S-adenosyl-l-methionine (AdoMet). AdoMet is a methyl donor in the methylation reaction, but its requirement for DNA cleavage remains unclear. In the present article we investigated MmeI interaction with DNA with the use of numerous methods. Our electrophoretic mobility shift assay revealed formation of two types of specific protein-DNA complexes. We speculate that faster migrating complex consists of one protein molecule and one DNA fragment whereas, slower migrating complex, which appears in the presence of AdoMet, may be a dimer or multimer form of MmeI interacting with specific DNA. Additionally, using spectrophotometric measurements we showed that in the presence of AdoMet, MmeI protein undergoes conformational changes. We think that such change in the enzyme structure, upon addition of AdoMet, may enhance its specific binding to DNA. In the absence of AdoMet MmeI binds DNA to the much lower extent.

Gene transfer of CuZn superoxide dismutase enhances the synthesis of vascular endothelial growth factor.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H2O2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H2O2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1.