Inhibition of Paracoccidioides lutzii Pb01 Isocitrate Lyase by the Natural Compound Argentilactone and Its Semi-Synthetic Derivatives

The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.     

Proteome characterization of Paracoccidioides lutzii conidia by using nanoUPLC-MSE

Fungi of the genus Paracoccidioides are the etiological agents of Paracoccidioidomycosis (PCM), the most prevalent mycosis in Latin America. Paracoccidioidomycosis infection is acquired by inhalation of Paracoccidioides conidia, which have first contact with the lungs and can subsequently spread to other organs/tissues. Until now, there have been no proteomic studies focusing on this infectious particle of Paracoccidioides. In order to identify the Paracoccidioides lutzii conidia proteome, conidia were produced and purified. Proteins were characterized by use of the nanoUPLC-MS^E approach. The strategy allowed us to identify a total of 242 proteins in P. lutzii conidia. In the conidia proteome, proteins were classified in functional categories such as protein synthesis, energy production, metabolism, cellular defense/virulence processes, as well as other processes that can be important for conidia survival. Through this analysis, a pool of ribosomal proteins was identified, which may be important for the initial processes of dimorphic transition. In addition, molecules related to energetic and metabolic processes were identified, suggesting a possible basal metabolism during this form of resistance of the fungus. In addition, adhesins and virulence factors were identified in the P. lutzii conidia proteome. Our results demonstrate the potential role that these molecules can play during early cell–host interaction processes, as well as the way in which these molecules are involved in environmental survival during this form of propagation.     

The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Paracoccidioidomycosis due to Paracoccidioides brasiliensis S1 associated with acquired immunodeficiency syndrome: A case report

BackgroundParacoccidioidomycosis (PCM) is an endemic disease in Latin America. In immunocompetent hosts, PCM occurs in two main clinical forms: acute and chronic. However, in HIV-infected patients PCM may show up simultaneous manifestations of acute and chronic forms.Case reportWe present the case of a patient diagnosed with HIV who had disseminated skin lesions and generalized lymphadenopathy, as well as respiratory and central nervous system involvement. The PCM diagnosis was confirmed by direct KOH examination, double immunodiffusion and the isolation of the fungus in samples of an abscess in the subcostal region. The isolate was identified as Paracoccidioides brasiliensis S1 by species-specific PCR using primers for protein-coding gene GP43 (exon 2) followed by PCR-RFLP of the alpha-tubulin gene.ConclusionsThere are few data in literature reporting species-specific molecular identification of Paracoccidioides in HIV/PCM patients. Therefore, this case report may contribute to improve the knowledge about this severe disease, its causative cryptic species, and its consequences to patients.     

Hepatic Disease with Portal Hypertension and Acute Juvenile Paracoccidioidomycosis: A Report of Two Cases and Literature Review

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole–trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.     

Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.     

Identification and characterization of Paracoccidioides lutzii proteins interacting with macrophages

Paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a systemic disorder that involves the lungs and other organs. The adherence of pathogenic microorganisms to host tissues is an essential event in the onset of colonization and spread. The host–pathogen interaction is a complex interplay between the defense mechanisms of the host and the efforts of pathogenic microorganisms to colonize it. Therefore, the identification of fungi proteins interacting with host proteins is an important step understanding the survival strategies of the fungus within the host. In this paper, we used affinity chromatography based on surface proteomics (ACSP) to investigate the interactions of pathogen proteins with host surface molecules. Paracoccidioides lutzii extracts enriched of surface proteins were captured by chromatographic resin, which was immobilized with macrophage cell surface proteins, and identified by mass spectrometry. A total of 215 proteins of P. lutzii were identified interacting with macrophage proteins. In silico analysis classified those proteins according to the presence of sites for N- and O-glycosylation and secretion by classical and non-classical pathways. Serine proteinase (SP) and fructose-1,6-bisphosphate aldolase (FBA) were identified in our proteomics analysis. Immunolocalization assay and flow cytometry both showed an increase in the expression of these two proteins during host–pathogen interaction.     

Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H. capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.     

Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.     

Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

Therapeutic treatment with scFv–PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM.     

Characterization of the Paracoccidioides Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus

Background

Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America.

Methodology/Principal Findings

In this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MS^E proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses.

Conclusions/Significance

In this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.     

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of Δ²⁴⁽²⁸⁾ sterol methyl reductase (SMR) and/or Δ⁽²⁴⁾-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.     

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.     

Saccharomyces cerevisiae Expressing Gp43 Protects Mice against Paracoccidioides brasiliensis Infection

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.     

Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.