Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

On the Cooperativity Effect in Watson and Crick and Wobble Pairs for a Halouracil Series and Its Potential Quantitative Application Studied through Surface-Enhanced Raman Spectroscopy.

The nature of the cooperativity effect of hydrogen bonds in Watson and Crick and wobble base pairs formed with thymine, uracil, and its 5-halogenated derivatives (5-fluoro, -chloro, and -bromouracil) has been studied through SERS and by using chemometric tools to process data and extract relevant information. Remarkable differences between the two kinds of pairs were clearly observed, and the behavior correlated to the withdrawing character of different substituents at the 5-position of uracil was verified. Multivariate analyses have also unveiled information about the pair's stability, and a stronger cooperativity effect seems to rule the Watson and Crick pairs when compared to wobble pairs. Defined patterns in the behavior of Watson and Crick pairs allowed the design of an indirect methodology for quantifying 5-bromouracil using a partial least squares (PLS) method with variable selection. Limit of detection (LOD) values of 0.037 and 0.112 mmol L-1 in the absence and presence of structurally similar interferences were reached, while its direct surface-enhanced Raman spectroscopy (SERS) quantification is only possible at ∼45 mmol L-1.

DPD functional tests in plasma, fresh saliva and dried saliva samples as predictors of 5-fluorouracil exposure and occurrence of drug-related severe toxicity.

OBJECTIVE: to evaluate plasma and salivary uracil (U) to dihydrouracil (UH2) ratios as tools for predicting 5-fluorouracil systemic exposure and drug-related severe toxicity, and clinically validate the use of dried saliva spots (DSS) as an alternative sampling strategy for dihydropyrimidine dehydrogenase (DPD) deficiency assessment. METHODS: Pre-chemotherapy plasma, fresh saliva and DSS samples were obtained from gastrointestinal patients (N = 40) for measurement of endogenous U and UH2 concentrations by LC-MS/MS. A second plasma sample collected during 5FU infusion was used for 5FU area under the curve (AUC) determination by HPLC-DAD. Data on toxicity was reported according to CTCAE. RESULTS: 15% of the patients developed severe 5FU-related toxicity, with neutropenia accounting for 67% of the cases. U, UH2 and [UH2,]/[U] were highly correlated between fresh and dried saliva samples (rs = 0.960; rs = 0.828; rs = 0.910, respectively). 5FU AUC ranged from 11.3 to 37.31 mg h L-1, with 46.2% of under-dosed and 10.3% over-dosed patients. The [UH2]/[U] ratios in plasma, fresh saliva and dried saliva samples were moderately correlated with 5FU AUC and adverse events grade, indicating a partial contribution of the variables to drug exposure (r = -0.412, rs = -0.373, rs = 0.377) and toxicity (r = -0.363, rs = -0.523, rs = 0.542). Metabolic ratios were lower in patients with severe toxicity (P < .01 salivary ratios, and P < .5 plasma ratios), and 5FU AUC were in average 47% higher in this group than in moderate toxicity. The diagnostic performance of [UH2]/[U] ratios in fresh saliva and DSS for the identification of patients with severe toxicity were comparable. CONCLUSIONS: The [UH2]/[U] metabolic ratios in plasma, fresh saliva and DSS were significantly associated with 5FU systemic exposure and toxicity degree. This study also demonstrated the applicability of DSS as alternative sampling for evaluating DPD activity.

Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula.

The cyanobacterial toxin cylindrospermopsin (CYN) has become a globally important secondary metabolite due to the negative effect it has on human and animal health. As a means of evaluating the risk of human exposure to CYN, the bioaccumulation and depuration of the toxin in lettuce (Lactuca sativa L.) and arugula (Eruca sativa Mill.) were investigated, after irrigation with contaminated water. The vegetables were irrigated for 7days with CYN (3, 5 and 10µg/L) contaminated water (bioaccumulation phase), and subsequently, irrigated for 7days with uncontaminated distilled water (depuration phase). In general, the bioaccumulation of CYN in both vegetables decreased with increasing exposure concentration. Bioconcentration factor (BCF) of CYN increased with the progression of the experiment at 3.0µg/L CYN, while the reverse occurred at 5 and 10µg/L CYN. In arugula, BCF increased at all CYN exposure concentrations throughout the study. The depuration of CYN decreased with increasing exposure concentration but was highest in the plants of both species with the highest bioaccumulation of CYN. Specifically, in plants previously irrigated with water contaminated with 3, 5 and 10µg/L CYN, the depuration of the toxin was 60.68, 27.67 and 18.52% for lettuce, and 47, 46.21 and 27.67% for arugula, respectively. Human health risks assessment revealed that the consumption of approximately 10 to 40g of vegetables per meal will expose children and adults to 1.00-6.00ng CYN/kg body mass for lettuce and 2.22-7.70ng CYN/kg body mass for arugula. The irrigation of lettuce and arugula with contaminated water containing low CYN concentrations constitutes a potential human exposure route.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.

A quantum biochemistry investigation of willardiine partial agonism in AMPA receptors.

We employ quantum biochemistry methods based on the Density Functional Theory (DFT) approach to unveil the detailed binding energy features of willardiines co-crystallized with the AMPA receptor. Our computational results demonstrate that the total binding energies of fluorine-willardiine (FW), hydrogen-willardiine (HW), bromine-willardiine (BrW) and iodine-willardiine (IW) to the iGluR2 ligand-pocket correlate with the agonist binding energies, whose experimental sequential data match our computational counterpart, excluding the HW case. We find that the main contributions to the total willardiine-iGluR2 binding energy are due to the amino acid residues in decreasing order Glu705 > Arg485 > Ser654 > Tyr450 > T655. Furthermore, Met708, which is positioned close to the 5-substituent, attracts HW and FW, but repels BrW and IW. Our results contribute significantly to an improved understanding of the willardiine-iGluR2 binding mechanisms.

Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15)N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

Apparent bioaccumulation of cylindrospermopsin and paralytic shellfish toxins by finfish in Lake Catemaco (Veracruz, Mexico).

Compared to the well-characterized health threats associated with contamination of fish and shellfish by algal toxins in marine fisheries, the toxicological relevance of the bioaccumulation of toxins from cyanobacteria (blue-green algae), as the primary toxigenic algae in freshwater systems, remains relatively unknown. Lake Catemaco (Veracruz, Mexico) is a small, tropical lake system specifically characterized by a year-round dominance of the known toxigenic cyanobacterial genus, Cylindrospermopsis, and by low, but detectable, levels of both a cyanobacterial hepatotoxin, cylindrospermopsin (CYN), and paralytic shellfish toxins (PSTs). In the present study, we evaluated, using enzyme-linked immunoassay (ELISA), levels of both toxins in several species of finfish caught and consumed locally in the region to investigate the bioaccumulation of, and possible health threats associated with, these toxins as potential foodborne contaminants. ELISA detected levels of both CYN and PSTs in fish tissues from the lake. Levels were generally low (≤ 1 ng g(-1) tissue); however, calculated bioaccumulation factors (BAFs) indicate that toxin levels exceed the rather low levels in the water column and, consequently, indicated bioaccumulation (BAF >1). A reasonable correlation was observed between measured bioaccumulation of CYN and PSTs, possibly indicating a mutual source of both toxins, and most likely cells of Cylindrospermopsis, the dominant cyanobacteria in the lake, and a known producer of both metabolites. The potential roles of trophic transport in the system, as well as possible implications for human health with regards to bioaccumulation, are discussed.

Biocatalysed halogenation of nucleobase analogues.

The synthesis of halogenated nucleosides and nucleobases is of interest due to their chemical and pharmacological applications. Herein, the enzymatic halogenation of nucleobases and analogues catalysed by microorganisms and by chloroperoxidase from Caldariomyces fumago has been studied. This latter enzyme catalysed the chlorination and bromination of indoline and uracil. Pseudomonas, Citrobacter, Aeromonas, Streptomyces, Xanthomonas, and Bacillus genera catalysed the chlorination and/or bromination of indole and indoline. Different products were obtained depending on the substrate, the biocatalyst and the halide used. In particular, 85% conversion from indole to 5-bromoindole was achieved using Streptomyces cetonii.

Influence of diet on oxidative DNA damage, uracil misincorporation and DNA repair capability.

The contribution of diet to cancer ranges from 10 to 80%. The low ingestion of antioxidants and enzymatic cofactors involved in DNA repair and methylation reactions and the high ingestion of chemical additives present in the modern diet, associated with genetic factors, could lead to genomic instability and the hypomethylation of proto-oncogenes, thus contributing to development of genetic-related diseases such as cancer. The present study evaluated the influence of diet on the level of oxidative DNA damage, misincorporated uracil and DNA repair capability in peripheral blood lymphocytes from two groups of individuals with antagonist diets as follows: (i) 49 healthy individuals with a diet rich in organic products, whole grains, fruit and vegetables and poor in processed foods (Group I) and (ii) 56 healthy individuals with diet rich in processed foods and poor in fruit and vegetables (Group II). Oxidative DNA damage, uracil incorporation and DNA repair capability were assessed by the comet assay. The individuals in Group I presented lower levels of oxidative DNA damage (oxidized purines and pyrimidines) and lower levels of DNA damage induced by ex vivo treatment with hydrogen peroxide (H(2)O(2)) than those individuals in Group II. The analysis of our results suggests that a diet rich in organic products, integral grains, fruit and vegetables and poor in industrialized products can protect against oxidative DNA damage and DNA damage induced by H(2)O(2).

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation.

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO2 production and O2 consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

First evidence of "paralytic shellfish toxins" and cylindrospermopsin in a Mexican freshwater system, Lago Catemaco, and apparent bioaccumulation of the toxins in "tegogolo" snails (Pomacea patula catemacensis).

Exposure to cyanobacterial toxins in freshwater systems, including both direct (e.g., drinking water) and indirect (e.g., bioaccumulation in food webs) routes, is emerging as a potentially significant threat to human health. We investigated cyanobacterial toxins, specifically cylindrospermopsin (CYN), the microcystins (MCYST) and the "paralytic shellfish toxins" (PST), in Lago Catemaco (Veracruz, Mexico). Lago Catemaco is a tropical lake dominated by Cylindrospermopsis, specifically identified as Cylindrospermopsis catemaco and Cylindrospermopsis philippinensis, and characterized by an abundant, endemic species of snail (Pomacea patula catemacensis), known as "tegogolos," that is both consumed locally and commercially important. Samples of water, including dissolved and particulate fractions, as well as extracts of tegogolos, were screened using highly specific and sensitive ELISA. ELISA identified CYN and PST at low concentrations in only one sample of seston; however, both toxins were detected at appreciable quantities in tegogolos. Calculated bioaccumulation factors (BAF) support bioaccumulation of both toxins in tegogolos. The presence of CYN in the phytoplankton was further confirmed by HPLC-UV and LC-MS, following concentration and extraction of algal cells, but the toxin could not be confirmed by these methods in tegogolos. These data represent the first published evidence for CYN and the PST in Lago Catemaco and, indeed, for any freshwater system in Mexico. Identification of the apparent bioaccumulation of these toxins in tegogolos may suggest the need to further our understanding of the transfer of cyanobacterial toxins in freshwater food webs as it relates to human health.

Thymine hydroperoxide as a potential source of singlet molecular oxygen in DNA.

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O2 (1Deltag)] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O2 (1Deltag). Spectroscopic evidence for generation of O2 (1Deltag) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D2O enhancement of O2 (1Deltag) production and the quenching effect of NaN3. Moreover, the presence of O2 (1Deltag) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O2 (1Deltag) derived from the H2O2/HOCl system and from the thermolysis of the N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O2 (1Deltag) generation was obtained by chemical trapping of O2 (1Deltag) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce4+ ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O2 (1Deltag) by the Russell mechanism and point to the involvement of O2 (1Deltag) in thymidine hydroperoxide cytotoxicity.

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains.

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.

Genetic interactions of the Aspergillus nidulans atmAATM homolog with different components of the DNA damage response pathway.

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for DeltaatmA, DeltauvsB, DeltachkA, and DeltachkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APC1), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Control of ribosome turnover during growth of the haloalkaliphilic archaeon Natronococcus occultus.

The metabolism of ribosomes during growth of the haloalkaliphilic archaeon Natronococcus occultus was examined. The ribosome content was higher during exponential growth and diminished to 35% of the maximum in the stationary stage. The incorporation of H3-orotic acid and C14-uracil into rRNA was higher during exponential growth. After that, it decreased to 39% of the maximum in the stationary stage. The labeling of non-ribosomal RNA took place almost exclusively in the exponential stage. From loss of radioactivity, the half-life of rRNA was 11.43, 14.85, 5.28 and 7.14 h during the initial, exponential, late exponential and stationary growth stages, respectively. These results suggested that increased synthesis combined with diminished degradation were responsible for the high ribosome content displayed by Ncc. occultus during exponential growth. In contrast, diminished synthesis together with increased degradation provoked its posterior loss.

A putative RNA editing from U to C in a mouse mitochondrial transcript.

Recently, we isolated and characterized a new mouse mitochondrial RNA molecule containing the mitochondrial 16S RNA plus 121 nt joined to the 5' end of the RNA. This fragment arises from the L strand of the same gene and we have named this transcript chimeric RNA. At position 121 of the RNA there is a C, which, according to the sequence of the mitochondrial 16S RNA gene, should be a U. We hypothesized that this RNA is synthesized having a U at position 121, which is later substituted to a C by a putative editing reaction. Based on the presence of sites for the restriction endonucleases RsaI and Fnu4HI around position 121, both forms of the RNA were detected in mouse tissues. To confirm the presence of the non-edited and putative edited RNA, a fragment containing the first 154 nt of the RNA was amplified by RT-PCR and cloned. The substitution of U for C was demonstrated by sequencing these clones. In vitro transcription experiments demonstrated that the substitution of U for C is not due to artifact of amplification or cloning. Moreover, in mitochondria from testis only the non-edited form was found. This, together with other experimental evidence, demonstrated that the base substitution was not due to polymorphism of the mitochondrial 16S RNA gene. This is the first demonstration of a substitution reaction from U to C in a mammalian mitochondrial transcript.

Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis.

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.