Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions

Philippe Totté; Tiffany Bonnefois; Lucia Manso-Silván

doi:10.1371/journal.pone.0305851

. 2024 Jun 27;19(6):e0305851. doi: 10.1371/journal.pone.0305851

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions

Philippe Totté ^1,^*, Tiffany Bonnefois ¹, Lucia Manso-Silván ¹

Editor: Rohana P Dassanayake²

PMCID: PMC11210856 PMID: 38935768

Abstract

We investigated the interactions of unopsonized and opsonized Mycoplasma mycoides subsp. mycoides (Mmm) with bovine macrophages in vitro. Mmm survived and proliferated extracellularly on bovine macrophage cell layers in the absence of Mmm-specific antisera. Bovine complement used at non-bactericidal concentrations did neither have opsonizing effect nor promoted intracellular survival, whereas Mmm-specific antisera substantially increased phagocytosis and Mmm killing. A phagocytosis-independent uptake of Mmm by macrophages occurred at a high multiplicity of infection, also found to induce the production of TNF, and both responses were unaffected by non-bactericidal doses of bovine complement. Bovine complement used at higher doses killed Mmm in cell-free cultures and completely abrogated TNF responses by macrophages. These results provide a framework to identify Mmm antigens involved in interactions with macrophages and targeted by potentially protective antibodies and point towards a pivotal role of complement in the control of inflammatory responses in contagious bovine pleuropneumonia.

Introduction

Mycoplasma mycoides subsp. mycoides (Mmm) is a wall-less bacterium and the causative agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease of cattle notifiable to the World Organization for Animal Health [1]. CBPP lung lesions and chronic carrier state are suggestive of an uncontrolled inflammatory response by the host’s immune system and immune evasion mechanisms developed by Mmm respectively [2]. Although macrophages patrolling the surface of respiratory mucosae represent the first line of defense against pathogens invading the lung, little is known of the interactions of these cells with Mmm. Macrophages containing Mmm antigens are found in the lungs of infected animals [3] and they produce the archetypal pro-inflammatory cytokine TNF upon infection in vitro [4]. However, studies investigating the fate of Mmm in the presence of macrophages are lacking.

Another major contributor to the host defense against infection is the complement system. Complement proteins are present in extracellular fluids allowing direct killing and non-specific opsonization, followed by increased phagocytosis of pathogens [5]. Non-specific opsonization can result in killing but also in intracellular survival if pathogens are able to prevent fusion of phagosomes with lysosomes [6, 7]. Upon activation, the complement promotes inflammation through the induction of pro-inflammatory cytokines [8, 9]. These pleiotropic effects of complement are not exclusive and may be dose-dependent. For example, the complement membrane attack complex responsible for the pathogen killing activity of complement can also induce the production of pro-inflammatory cytokines by phagocytes when used at non-bactericidal doses [9]. Thus, in CBPP, interactions between Mmm, macrophages and complement may either benefit the host, through direct or opsonization-mediated killing of Mmm or: paradoxically; harm the host, through opsonization-mediated intracellular survival of Mmm and amplification of inflammatory responses.

Although previous work suggests that mycoplasmas are generally phagocytosed by macrophages only in the presence of specific antiserum [10], the question remains open for Mmm. Also, effects of complement other than opsonization, such as direct killing and cytokine production, may impact disease outcome in CBPP. Therefore, the following study was undertaken to investigate the interaction of bovine macrophages with Mmm under physiological conditions, i.e., in the presence of complement prepared from the same cell donor. Firstly, the effect of bovine complement on the viability of Mmm was analyzed to determine non-bactericidal concentrations. Secondly, the uptake of Mmm by macrophages and subsequent extra- and intracellular survival in the presence of bovine complement at non-bactericidal doses was investigated. Finally, we addressed the possibility that complement acts as a potentiator of Mmm-induced TNF responses of macrophages.

Methods

Bacterial strains

Non-fluorescent Mmm strain 8740-Rita and Mycoplasma bovis strain Oger2, and fluorescent mNeonGreen-expressing Mmm mutant strain 8740-Rita clone 6 (RN6), were grown in PPLO-based medium as described previously [11]. Mycoplasmas obtained from log-phase cultures were washed once in PBS by centrifugation at 10000 x g for 10 min before resuspension in macrophage culture medium (see below) at 1-2x10⁹ colony forming units per mL, or CFU/ml, as determined through dilution and plating on PPLO-based media supplemented with 1% Noble Agar (Difco, USA). Briefly, serial 10-fold dilutions in PBS were prepared in a final volume of 500μL with a change of tip and vortexing between each tube. 20μL from each tube were spotted onto PPLO agar plates and incubated at 37°C for four days before counting the colonies under a light microscope.

Bovine complement and antiserum

Non-decomplemented bovine sera obtained from 3 adult Jersey cattle housed at Cirad’s animal facility served as a source of complement. These animals were purchased in France were CBPP has been eradicated and were kept outdoor after approval by the Languedoc-Roussillon regional ethics committee (French CE-LR#36) in the authorized project using animals for scientific purposesAPAFIS#27628. Sera were aseptically collected from whole blood tubes after clotting for 30 min at room temperature at 19–23°C, followed by centrifugation at 800 x g for 20 min. Single-use aliquots of non-decomplemented sera were kept in liquid nitrogen until use after quick thawing at 37°C. Bactericidal capacities of bovine complement were assessed by incubation of 1x10⁶-1x10⁸ viable Mmm with different concentrations of non-decomplemented bovine sera at 100 μL/well in 96-well flat-bottom-plates for 1 h at 37°C and 5% CO₂ before vortexing and CFU titration. That range of viable Mmm corresponds to the lowest and highest number of bacteria used to infect macrophages. The specific Mmm antiserum was obtained by pooling sera from two-years old Zebu cattle recovering from an experimental infection with Mmm strain 8740-Rita [12]. These animals were selected on the basis of their positivity using a CBPP-specific cELISA [13] and a record of clinical signs typical of CBPP. Prior to use, Mmm antisera were decomplemented by heating at 56°C for 30min.

Macrophages and bacterial infection

Macrophages were derived from monocytes obtained from the same three animals used to prepare non-decomplemented bovine sera. Briefly, monocytes were purified from PBMC by positive selection of CD14+ cells using anti-human CD14 magnetic beads (Miltenyi, Germany) according to the manufacturer’s instructions. Monocytes were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 0.4 mg/mL ampicillin and 10% heat-inactivated fetal calf serum (FCS; Eurobio AbCys, France), seeded at 3 x 10⁵cells/well of 96-well flat-bottom-plates in 200 μl, and incubated for 6 days at 37°C and 5% CO₂ with a change of half of the medium at day 3. Before infection, the medium was removed and replaced with IMDM lacking FCS to which the following stimuli were added to reach a final volume of 100 μl/well: i) appropriate amounts of mycoplasmas to obtain MOIs of 1, 10 and 100; and ii) non-decomplemented autologous bovine sera or antiserum or FCS. Non-decomplemented bovine sera were used at non-bactericidal doses ranging from 5 to 10% v/v depending on the donor, whereas antiserum was used at 20% v/v and FCS at 10% v/v. After 1 h incubation, the medium was replaced and 100μl of complete IMDM medium was added to the wells before harvesting or further incubation. For harvesting macrophages, monolayers were scrapped off using 200μl tips, pelleted by centrifugation at 500 x g for 5 min, resuspended in 200 μL PBS, and passaged 5 times through a 27gauge needle, which lysed more than 80% of cells as shown by Trypan blue viability counts. Mmm CFU titers were determined as described above. For gentamycin assays, an MOI of 10 was used, as it was shown in preliminary macrophage-free assays that addition of 400 μg/ml of gentamicin to the culture for 3 hours reliably killed 100% of mycoplasmas. Cultures infected with M. bovis strain Oger2 were used as positive controls of intracellular survival in the presence of gentamycin [14].

Flow cytometry

Macrophages were infected with fluorescent and non-fluorescent Mmm at MOIs of 500–1000 as described above. Due to the high MOI, non-decomplemented bovine sera could be used at up to 40% v/v without affecting Mmm viability. Before harvesting, the medium was replaced by 100 μl/well of fresh medium and cells were scraped off, homogenized by pipetting up and down, and transferred to 5 ml cytometry tubes. After addition of 10 μL/tube of propidium iodide (PI, BD Biosciences, USA) and 10min incubation at 4°C in the dark, 100 μL of sheath fluid was added to each tube. Preliminary macrophage-free tests showed that 100% of Mmm took up PI in these conditions. Since PI and mNeonGreen fluoresce in different wavelength, it was possible to gate out PI positive macrophages to exclude dead cells (i.e., with loss of membrane integrity) and extracellular Mmm. This allowed analysis of strictly intracellular mNeonGreen-dependent fluorescence. CytochalasinD (Sigma, France), an inhibitor of actin-dependent phagocytosis, was used at 10 μg/ml. At least 5000 events were analyzed within the PI negative gate with a FacsCanto flow cytometer (BD Biosciences) equipped with the FacsDiva software (BD Biosciences). Gates and quadrants were used to delineate populations of interest and calculate statistics respectively. Results were expressed in percentages of cells fluorescing above background (i.e., fluorescence produced by macrophages infected with non-fluorescent Mmm) and geometric mean fluorescence intensity (MFI), which is proportionally related to numbers of mNeonGreen+ Mmm per cell.

ELISA

Supernatants were collected from macrophage cultures infected as above for 24 h with Mmm at MOIs ranging from 100 to 1000 and stored at -20°C until use. Non-decomplemented bovine sera were used at both bactericidal and non-bactericidal concentrations and the highest dose was also used for FCS. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Invivogen, France), used at 2 μg/ml, served as a positive control. To quantify bovine TNF, a sandwich ELISA was performed using a matched antibody pair (BioRad, France) as previously described [15]. Supernatants were tested in duplicates and results obtained from a labsystems multiskan MS ELISA reader were expressed in mean optical densities.

Statistics

A non-parametric Mann–Whitney U-test (https://www.socscistatistics.com/tests/mannwhitney/) was used to analyze differences between responses obtained with various stimuli. Single and double asterisks represent p values of <0.05 and <0.01 respectively. A difference was considered to be significant at a p value of <0.05.

Results

Bactericidal effect of bovine complement on Mmm viability in the absence of macrophages

A dose-dependent effect of non-decomplemented bovine sera, containing bovine complement, on Mmm CFU titers was observed in vitro (Fig 1A). Depending on the dose and animals used the decrease in CFU titers varied between 0 and 3 logs, indicating a potent effect of bovine sera and a high variability among animals. There was no sign of Mmm aggregation induced by non-decomplemented serum when cultures were checked under a light microscope, and passage through a 27G needle had no impact on CFU titers, indicating that reduced viability occurred through direct cytotoxicity. The killing effect was due to complement since it was neutralized by heating sera at 56°C for 30 min (Fig 1B) a method known to inactivate complement [5]. The mycoplasmacidal activity and inter-animal variability of bovine sera remained consistent throughout the study (S1 Fig).

Effect of bovine complement on Mmm opsonization and survival in the presence of macrophages

A protocol was used to focus on Mmm associated to macrophages and to exclude non-adherent and plastic-adherent mycoplasmas from the analysis (see methods). Furthermore, no cytopathological effects of Mmm on macrophage monolayers were detected at any MOI used. In the absence of bovine complement and antiserum, complete survival of Mmm occurred at 24 h and growth of up to 1 log was observed at 48h, indicating no killing activity by macrophages (Fig 2). Similar trends were observed at MOIs of 1 and 100 whereas only survival of Mmm without growth (i.e., CFU titers remained constant) was observed in the absence of macrophages (S2 Fig). The addition of non-decomplemented bovine sera at non-bactericidal concentrations had no effect on Mmm viability at any time-points (Fig 2). As expected, addition of Mmm-specific antiserum induced significant killing activity after 24 h and up to 2.5 logs decrease in CFU titers at 48h (Fig 2).

Fig 2 — Macrophages were infected with *Mmm* at an MOI of 10 in the absence (FCS) or presence (serum) of non-decomplemented bovine sera at non-bactericidal concentrations and incubated for 1, 24, and 48h before assessment of macrophages-associated *Mmm* titers. Anti-*Mmm* antiserum (antiserum) was added to additional wells as positive controls of bactericidal activity. Results from 3 different experiments using 3 animals are shown as mean+/-SD of CFU expressed in log10. Asterisks indicate statistically significant differences with FCS.

In order to analyze intracellular survival of Mmm experiments were repeated in the presence of gentamicin to kill all extracellular mycoplasmas. This was performed only at an MOI of 10, which represented a concentration of 3x10⁶ CFU/ml of Mmm, since gentamicin did not kill 100% extracellular mycoplasmas at higher Mmm concentrations. As seen in Fig 3, gentamicin assays indicated that the overwhelming majority of Mmm survival at 24 h was due to extracellular mycoplasmas and that bovine complement had no effect on Mmm survival inside macrophages. This was not due to a bias in gentamicin assay since significant survival was observed with M. bovis Oger2 (Fig 3).

Fig 3 — Macrophages were infected with *Mmm* at an MOI of 10 in the absence (FCS) or presence (serum) of bovine complement at non-bactericidal concentrations and incubated for 24 h. Cells were then treated for 4-hours with gentamycin before harvesting and assessment of macrophage-associated *Mmm* titers. Wells containing macrophages infected with M. *bovis* (Oger2) were used as positive controls of intracellular survival in the presence of gentamycin. Results from 3 different experiments using 3 animals are shown as mean+/-SD of CFU expressed in log10.

Effect of bovine complement on the uptake of fluorescent Mmm by macrophages

We were able to analyze Mmm-dependent intracellular fluorescence by gating out extracellular fluorescence as explained in “Methods”. As seen in Fig 4, in the absence of bovine complement or antiserum, more than 90% of macrophages had engulfed fluorescent Mmm but at low levels as indicated by low FITC fluorescence intensity (“Li” in Fig 4A). Indeed, FITC signals did not exceed one log above background level and could not be detected when MOIs of 100 or less were used (data not shown). No differences were detected between 1 and 24 h PI in both percentages of positive cells and MFI (Fig 4A and 4B). Non-decomplemented bovine sera used at non-bactericidal concentrations had no effect on FITC fluorescent levels at any time-points (Fig 4A and 4B). Data at 48h were not exploitable due to high background fluorescence. The low-level uptake was not affected by cytochalasinD (“Li” in Fig 4C) indicating that it was not dependent on phagocytosis. A substantially stronger FITC fluorescence intensity was observed in the presence of antiserum indicating a strong increase in Mmm uptake by macrophages (“Hi” in Fig 4A). At 24 h PI, the % of cells within the “Hi” gate increased while the mean fluorescence intensity decreased (gate P5 and MFI in Fig 4B) suggesting partial destruction of fluorescent Mmm within macrophages. Moreover, the vast majority of highly fluorescent cells disappeared in the presence of cytochalasinD (Fig 4C) indicating that the uptake of Mmm by macrophages in the presence of antiserum was dependent on phagocytosis.

Fig 4 — Macrophages were infected with either non-fluorescent or fluorescent *Mmm* at an MOI of 500–1000, and either in the absence (FCS) or in the presence (serum) of non-decomplemented bovine sera at non-bactericidal concentrations. They were incubated for 1 h (a) and 24 h (b) before analysis of intracellular FITC fluorescence by flow cytometry (see Methods). Finally, the effect of cytochalasinD, an inhibitor of phagocytosis, on *Mmm* uptake by macrophages is shown in c). Anti-*Mmm* antiserum (antiserum) was added to additional wells as positive control of specific phagocytosis. Typical histogram plots are shown for one representative animal as an example. Histograms and arrows display background (bgd, hatched line), and low (Li, green line) and high (Hi, green line) fluorescence intensities produced by macrophages infected with non-fluorescent and fluorescent *Mmm* respectively. Percentages of highly fluorescent cells and MFI are given by the P5 interval.

The lack of effect by bovine complement on the uptake of Mmm by macrophages was confirmed for all three animals tested (Table 1). Indeed, the addition of non-decomplemented bovine sera at non-bactericidal concentrations had no impact on numbers of positive cells and MFI when compared to FCS at 1 h and 24 h PI. In addition, the positive effect of antiserum on Mmm uptake was confirmed as well as the increase in percentage of positive cells at 24 h PI accompanied by a decrease in MFI, indicating progressive destruction of phagocytized fluorescent Mmm.

Table 1. Effect of bovine complement (serum) from three different animals (mean+/-SD) on the uptake of fluorescent Mmm by autologous macrophages as measured by flow cytometry 1 h and 24 h post infection.

Stimuli	Low fluorescence intensity^(a)				High fluorescence intensity^(a)
	% pos ^(b)		MFI^I		% pos ^(b)		MFI^(c)
	1 h	24 h	1 h	24 h	1 h	24 h	1 h	24 h
FCS	93+/-2	96+/-5	1013+/-33	995+/-33	7+/-3	10+/-1	NA	NA
Serum	92+/-3	99+/-3	1008+/-52	1050+/-42	8+/-4	9+/-2	NA	NA
Antiserum^(d)	NA	NA	NA	NA	63+/-10	94+/-5	14518	6905
							+/-1313	+/-948

Open in a new tab

(a): low and high fluorescence intensities above background fluorescence (see also Fig 4)

(b): Percentages of positive celI(c): Mean fluorescence intensi ty

(d): sera from CBPP convalescent animals

NA: not applicable. The effect of antiserum is measurable only within the high fluorescence intensity whereas, in the presence of complement, the vast majority of positive cells were found in the low fluorescence intensity range.

Effect of bovine complement on Mmm-induced TNF production by macrophages

As shown in Fig 5, in the absence of bovine sera and antiserum, Mmm induced the production of TNF by macrophages when MOIs of 500–1000 were used. At an MOI of 100, TNF was not detected (not shown). There was no effect of bovine complement at non-bactericidal doses (Fig 5, serum1). In order to analyze the effect of non-decomplemented bovine sera at bactericidal doses, concentrations of both non-decomplemented bovine sera and FCS had to be increased. For one animal, a substantial increase in background (i.e., TNF induced by FCS alone in the absence of Mmm) prevented its use. Nevertheless, non-decomplemented bovine sera from the remaining two animals, used at bactericidal concentrations inducing at least 1log decrease in Mmm viability, almost completely abrogated the production of TNF induced by Mmm (Fig 5, serum2).

Discussion

Given their pivotal role in first-line immune defenses against mucosal pathogens, we have investigated the interactions between macrophages and Mmm, the causal agent of CBPP. Non-decomplemented bovine sera were used as a source of complement to mimic physiological conditions present at infected sites and cryopreservation in liquid nitrogen was instrumental in overcoming the instability of complement. The strong bactericidal effect of some non-decomplemented bovine sera on Mmm viability that we observed in this study is in conflict with previous work [16]. One likely explanation is that we used bovine sera instead of guinea pig sera. Similarly, non-decomplemented caprine sera was found to reduce the viability of Mycoplasma mycoides subsp. capri (Mmc), Mmm’s closest relative [17]. Given its stability over time in vivo and variable potency between animals, it would be interesting to assess whether a correlation exists between the anti-Mmm effect of bovine sera and the resistance of animals to CBPP. Considering the pleiotropic effects of complement on immune responses to pathogens, we have explored the possible impact of bovine complement, used at non-bactericidal concentrations, on important functions of macrophages such as non-specific opsonization-mediated phagocytosis and production of pro-inflammatory TNF.

In the first place, we analyzed the kinetics of Mmm survival in the presence of bovine macrophages and in the absence of complement and antiserum. Lysis of macrophages prior to Mmm titration allowed taking into account both intracellular and membrane-associated mycoplasmas. Mmm CFU titers remained stable after 24 h and increased by up to 1log after 48h, indicating the absence of measurable killing by macrophages. Little or negligible numbers of macrophage-associated Mmm survived intracellularly in the presence of gentamicin, suggesting that viable Mmm were mainly located on cell membranes. Absence of killing by macrophages or even growth on macrophages in the absence of specific antiserum and complement has been reported previously for other mycoplasmas [10, 18]. We found no measurable effect of bovine complement on Mmm CFU titers, suggesting that neither non-specific opsonization-mediated killing nor intracellular survival occurred under our experimental conditions. It should be noted that pre-incubation of Mmm with bovine complement for 30 min at 37°C before infecting macrophages rather than concomitantly did not change the results. However, we cannot exclude the possibility that the presence of Mmm growing on macrophages masks a low-level killing activity. On the other hand, Mmm titers substantially decreased over time when anti-Mmm antiserum was added to the medium indicating that intrinsic killing capacities of macrophages were not altered. Again, absence of killing by macrophages, even in the presence of complement, has been shown previously for several mycoplasmas [10]. It is possible that the presence of a polysaccharide capsule [19] protects non-opsonized and non-specifically opsonized Mmm from phagocytosis [20] which may be further investigated using non capsulated variants [16].

In order to gain a better insight on the interactions of Mmm with bovine macrophages, we used a more direct approach based on flow cytometry-assisted monitoring of intracellular fluorescence of phagocytes after infection with fluorescent Mmm. Our results show that, in the absence of complement and antiserum, more than 90% of macrophages take up fluorescent Mmm at low levels, as indicated by low mean fluorescence intensities. Unfortunately, in macrophages-free cultures, gentamicin was not 100% bactericidal at the Mmm concentrations used for flow cytometry.Thus, preventing assessment of the viability of intracellular fluorescent Mmm. The lack of decrease in fluorescence over time suggests that either no killing occurred or that it was not sufficient to cope with ongoing growth of Mmm on macrophages. CytochalasinD had no effect on the number of cells harboring low intracellular fluorescence, suggesting that the uptake of Mmm by macrophages was independent of phagocytosis. Interestingly, it has recently been shown that Mmc interacts with C-type lectin receptors (CTLRs) [21] some of which mediate endocytosis. Several CTLRs are present on macrophages, which warrants further studies on their potential role in CBPP. Moreover, pathogen-associated molecular patterns (PAMPs) of Mmm recognized by these receptors have potential as virulent factors. Bovine complement used at non-bactericidal concentrations had no effect on the uptake of Mmm by macrophages at any time points, confirming viability experiments using CFU titration. On the other hand, anti-Mmm antiserum induced a strong and phagocytosis-dependent increase in Mmm uptake leading to more than 90% of cells located in the high fluorescence intensity gate at 24 hpi. Moreover, the mean fluorescence intensity within that gate decreased at 24 hpi confirming the progressive destruction of Mmm as seen with CFU titration. These last results are intriguing since Mmm and related mycoplasmas possess a two-protein system capable of cleaving immunoglobulins G (IgG) [22]. Decomplementation of anti-Mmm antisera did not affect their opsonizing potency indicating that IgGs are most likely involved.

Our results confirm previous work demonstrating the capacity of Mmm to induce TNF production by alveolar macrophages in the absence of either complement or antiserum (Jungi et al., 1996). Our results show that the use of peripheral blood-derived MDMs represents a valid alternative to alveolar macrophages, whose preparation requires greater expertise and has a greater impact on animal welfare. Interestingly, TNF was induced only at high MOIs (i.e., >500) also shown by flow cytometry to be associated with phagocytosis-independent engulfment of Mmm. This suggests a possible involvement of autophagy in the pro-inflammatory response of macrophages to Mmm. There was no beneficial effect of bovine complement on the TNF response of infected macrophages when used at non-bactericidal concentrations. The effect of bovine complement at bactericidal doses was also analyzed, considering the possible release of pro-inflammatory molecules from Mmm undergoing lysis. We found that, when used at bactericidal concentrations, bovine complement efficiently abrogated the production of TNF by macrophages in response to Mmm infection.

In summary, using a combination of CFU titration, gentamicin assays, fluorescent Mmm, and TNF ELISA, we were able to show that: i) macrophages were not capable of killing Mmm efficiently in the absence of antiserum; ii) bovine complement used at non-bactericidal doses neither had opsonizing effect nor did it promote intracellular survival; iii) bovine complement did not impact the phagocytosis-independent Mmm uptake by macrophages that occurred in the absence of antiserum; iv) bovine complement used at non bactericidal concentrations did not amplify Mmm-induced TNF responses of macrophages, and even abrogated that response when used at concentrations inducing bactericidal activity. These results provide a framework to identify PAMPs as well as Mmm antigens recognized by CTLRs and protective antibodies. In addition, they point towards a role for complement in the control of TNF-dependent inflammatory responses in CBPP.

Supporting information

S1 Fig. Stability of the bactericidal effect of bovine complement.

(TIF)

pone.0305851.s001.tif^{(119.9KB, tif)}

S2 Fig. Survival of Mmm in vitro in supplemented IMDM medium in the absence of macrophages.

(TIF)

pone.0305851.s002.tif^{(95.7KB, tif)}

Data Availability

Data are available online at https://zenodo.org/record/7442582#.ZAiYrHbMJOC.

Funding Statement

The author(s) received no specific funding for this work.

References

1.World Organization for animal Health (2015). https://www.woah.org/en/what-we-do/animal-health-and-welfare/animal-diseases/?_alphabet=C. Accessed 20 Sept 2022
2.Provost A, Perreau P, Bréard A. Contagious bovine pleuropneumonia (1987) Rev Sci Tech Off. int. Epiz., 6 (3), 625–679. 10.20506/rst.6.3.306 [DOI] [PubMed] [Google Scholar]
3.Scanziani E, Paltrinieri S, Boldini M, Grieco V, Monaci C, Giusti AM, et al. (1997) Histological and immunohistochemical findings in thoracic lymph nodes of cattle with contagious bovine pleuropneumonia. J Comp Pathol. Aug;117(2):127–36. doi: 10.1016/s0021-9975(97)80029-1 [DOI] [PubMed] [Google Scholar]
4.Jungi TW, Krampe M, Sileghem M, Griot C, Nicolet J (1996) Differential and strain-specific triggering of bovine alveolar macrophage effector functions by mycoplasmas. Microb Pathog. 2:487–498. doi: 10.1006/mpat.1996.0078 [DOI] [PubMed] [Google Scholar]
5.Volanakis J.E. (1998) Overview of the complement system, in: Volanakis J.E., Frank M.M., (Eds.), The human complement system in health and disease, Marcel Dekker Inc., New York, 1998, pp. 9–32. doi: 10.1201/9780367800772 [DOI] [Google Scholar]
6.Cosma CL, Sherman DR, Ramakrishnan L. (2003) The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol 57:641–76. doi: 10.1146/annurev.micro.57.030502.091033 [DOI] [PubMed] [Google Scholar]
7.Horwitz M. A. (1983) The Legion’aires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108–2126. 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Okusawa S, Yancey KB, van der Meer JW, Endres S, Lonnemann G, Hefter K, et al. (1988) C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha. J Exp Med. 1;168(1):443–8. doi: 10.1084/jem.168.1.443 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Laudisi F, Spreafico R, Evrard M, Hughes TR, Mandriani B, Kandasamy M, et al. (2013) Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1β release. J.Immunol. Aug 1;191(3):1006–10. 10.4049/jimmunol.1300489 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Marshall AJ, Miles RJ, Richards L. (1995) The phagocytosis of mycoplasmas. J Med Microbiol. Oct;43(4):239–50. doi: 10.1099/00222615-43-4-239 [DOI] [PubMed] [Google Scholar]
11.Bonnefois T, Vernerey MS, Rodrigues V, Totté P, Puech C, Ripoll C, et al. (2016) Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades. J Biotechnol. Oct 20;236:35–44. doi: 10.1016/j.jbiotec.2016.08.006 [DOI] [PubMed] [Google Scholar]
12.Yaya A, Golsia R, Hamadou B, Amaro A, Thiaucourt F. Essai comparatif d’efficacité des deux souches vaccinales T1/44 et T1sr contre la péripneumonie contagieuse bovin. Rev Elev Med Vet Pays Trop. 1999;52(3–4):171–179. https://www.vetres.org/articles/vetres/pdf/2006/05/v6049.pdf [Google Scholar]
13.Le Goff C., Thiaucourt F., 1998. A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP). Vet. Microbiol., 60: 179–191. https://www.sciencedirect.com/science/article/pii/S0378113598001564 doi: 10.1016/s0378-1135(98)00156-4 [DOI] [PubMed] [Google Scholar]
14.Suleman M, Prysliak T, Clarke K, Burrage P, Windeyer C, Perez-Casal J. (2016) Mycoplasma bovis isolates recovered from cattle and bison (Bison bison) show differential in vitro effects on PBMC proliferation, alveolar macrophage apoptosis and invasion of epithelial and immune cells. Vet Microbiol. Apr 15;186:28–36. 10.1016/j.vetmic.2016.02.016 [DOI] [PubMed] [Google Scholar]
15.Totté P, Puech C, Rodrigues V, Bertin C, Manso-Silvan L, Thiaucourt F. (2015) Free exopolysaccharide from Mycoplasma mycoides subsp. mycoides possesses anti-inflammatory properties. Vet Res. Oct 21;46:122. doi: 10.1186/s13567-015-0252-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Gaurivaud P, Lakhdar L, Le Grand D, Poumarat F, Tardy F. (2014) Comparison of in vivo and in vitro properties of capsulated and noncapsulated variants of Mycoplasma mycoides subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine pleuropneumonia. FEMS Microbiol Lett. Oct;359(1):42–9. 10.1111/1574-6968 [DOI] [PubMed] [Google Scholar]
17.Schieck E, Lartigue C, Frey J, Vozza N, Hegermann J, Miller RA, et al. (2016) Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance. Mol Microbiol. Jan;99(1):55–70. 10.1111/mmi.13213. [DOI] [PubMed] [Google Scholar]
18.Howard CJ, Taylor G, Collins J, Gourlay RN. (1976) Interaction of Mycoplasma dispar and Mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leukocytes. Infect Immun. Jul;14(1):11–7. doi: 10.1128/iai.14.1.11-17.1976 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Buttery S.H. (1970) Hapten inhibition of the reaction between Mycoplasma mycoides polysaccharide and bovine antisera. Immunochemistry, 7, 305–310. doi: 10.1016/0019-2791(70)90168-0 [DOI] [PubMed] [Google Scholar]
20.Weis J.J., Law S.K., Levine R.P., Cleary P.P., (1985) Resistance to phagocytosis by group A streptococci: Failure of deposited complement opsonins to interact with cellular receptors, J. Immunol. 134 (1985) 500–505. [PubMed] [Google Scholar]
21.Lindenwald DL, Monteiro JT, Rautenschlein S, Meens J, Jung K, Becker SC, et al. (2020) Ovine C-type lectin receptor hFc-fusion protein li–rary—A novel platform to screen for host-pathogen interactions. Vet Immunol Immunopathol. Apr 18;224–230. 10.1016/j.vetimm.2020.110047 [DOI] [PubMed] [Google Scholar]
22.Arfi Y, Minder L, Di Primo C, Le Roy A, Ebel C, Coquet L, et al. (2016) MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G. Proc Natl Acad Sci U S A. May 10;113(19):5406–11. doi: 10.1073/pnas.1600546113 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0305851.r001

Decision Letter 0

Jianhong Zhou

10 Jan 2024

PONE-D-23-32412Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditionsPLOS ONE

Dear Dr. Totté,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jianhong Zhou

Staff Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [the article was preprinted and recommended by PCI https://microbiol.peercommunityin.org/articles/rec?id=5] Please clarify whether publication was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to authors

In the manuscript by Totté et al., the authors have described interactions of Mycoplasma mycoides subsp. mycoides (Mmm) with bovine macrophages using in vitro killing and phagocytosis assays. While the manuscript focuses on an essential area of research of Contagious Bovine Pleuropneumonia by conducting experiments to identify interaction of macrophages (from the target species) with Mmm, the data reported in the manuscript is not enough to support the current conclusions. Moreover, the authors have made various highly speculative statements without providing any evidence whatsoever to in the manuscript.

Unfortunately, in its current form the manuscript cannot be accepted. The authors must provide further experimental data in order to consider the publication of the manuscript. These are mentioned below.

Line 108-109: In this manuscript, the methodology used to study the interaction of Mmm and macrophages is extremely important. So, Mmms were left with the macrophages for 1 hr in all the experiments? Were the cells washed before adding different serums.

The way methodology is written currently, it seems that both mycoplasmas and serums were added together. It is very important for the results observed in Fig 2 because if the mycoplasmas and serums were added together then its really not different compared to the data presented in Fig 1.

The authors need to clarify this.

Flow cytometry analysis (Line 130-132): The flow cytometry analyses are important in order to understand whether Mmms are internalised (gone inside of the macrophages) or associated (made complex and stuck outside) with macrophages. Based-on the current gating strategy how would you differentiate macrophage-associated Mmm from macrophage-internalised Mmm?

Given that Mmms are so small, it will be very challenging to differentiate the macrophage-associated Mmm from macrophage-internalised Mmm. Due to this limitation, both the externally bound Mmm (large proportion) and internalised (minimum proportion) to macrophages will generate the same double positive signal (FITC + PI). Therefore, to understand what is going on here the side scatter and forward scatter graphs must be presented. In addition, the gating strategy must be presented in the supplementary information.

Line 180: Based on the methodology, it doesn't see that the authors have focused on Mmm associated to the macrophages. Please clarify.

Line 181: The authors mention that "no cytopathological effects were detected on macrophages". Did the authors measure viability of the macrophages?

Line 186: This is bit confusing. MOI is always represented in relation to cells, but the authors are saying in the absence of macrophages. Please clarify. The data must be presented for MOI 1 and 100 even thought, these MOI showed a similar trend.

Line 205: How was this conclusion made? The authors just mentioned that that "overwhelming majority of Mmm survival at 24 h was due to extracellular mycoplasmas". I don't think that the authors can make this conclusion based on the current results. First, it needs to be demonstrated that Mmm can survive in macrophages. Given that very low number of Mmm were detected in the G+ treatment group, it is almost impossible to say that "complement had no effect on Mmm survival inside macrophages".

Line 212: Fig 3 Given that the authors performed experiments to assess intracellular survival of Mmm in macrophages, this should be macrophages-internalised and not macrophages-associated.

Line 220: These results are contradictory to the previous observations presented in Fig 3. In that, hardly any Mms were internalised but here the authors claim that over 90% were engulfed by the macrophages. Please clarify these contradicting results.

Also, proper gating strategy must be presented in the supplementary information including side and forward plots, gating for any possible clumps of neogreen mutant strain.

What was the rational of using 500-1000 MOI of Mmm in these experiments? In the previous Fig. the authors have mentioned that gentamicin did not kill Mmm at MOI higher than 100, indicating that Mmm higher than MOI of 100 are likely to associated with macrophages and NOT internalised. Please describe these discrepancies in your results.

Line 222: While it is understandable the rational of using neogreen expressing strain, I think there is bigger fundamental problem here. Given that no FITC signal was detected when MOI of 100 or less were used, it clearly indicates that both the WT and neogreen expressing strains are not behaving the same when it comes to their ability to infect or engulfed by macrophages. The first question, which must be answered that how comparable these two different strains are in terms of their ability to internalise or engulfed by macrophages. As far as I see, there is no data on this. Therefore, no conclusion can be made from any comparisons of the mutant and WT Mmm strains in this study.

Line 226: Given that the uptake (internalisation) is low, this conclusion cannot be made.

Line 242: Fig 4 By looking at the graphs, background fluorescence levels (hatched line) appear to be changing. Moreover, it should be at "zero" and not 10^2 or 10^3. It seems like that flow cytometry gating wasn't done properly and the gating strategy must be presented as the supplementary material.

Line 275: Mmm were diluted in PBS. Can the authors provide the source of PBS and other reagents used in the cell-culture experiments? Given that TNF levels can be easily induced even by a small amount of co-contaminating endotoxin levels in reagents, it is absolutely important to use certified endotoxin free reagents in cell culture experiments.

Line 278-279: I don't think, that is the correct way of reporting experimental findings. That's the variability of the outbred animals. You can't just take two best readings out of three. The results from the third animal must be reported. Otherwise, this experiment becomes, redundant.

Line 292: Fig 5 so, 2 animals each for serum 1 and serum 2? Is that correct?

Line 315: Yes, this is a most likely scenario, but authors kept saying that Mmms have internalised. So, results need to be updated.

Line 319: The authors should avoid using "internalisation or intracellular" because based on the results presented these conclusions can't be made.

The current flow cytometry analyses are not sufficient to conclude that Mmms are internalised into macrophages. Other methodology such as confocal microscopy could be used to analyse internalisation of Mmm into macrophages.

Line 333: Please see the comment for flow cytometry analysis.

Line 338: Again, please see the comment for line 220.

Line 345: But the authors just mentioned (in line 339) that the uptake of Mmm was independent of phagocytosis. Please clarify and discuss these in the discussion section.

Line 346: Again, the intensity can also increase if Mmm + macrophages form a complex and not necessarily internalised. So, the gating strategy must be presented with side and forward scatter graphs.

Line 352: Why did the authors not comment on the third animal, which showed substantial increase in TNF levels. The impact of that on the study should be discussed here.

Line 356: There are no evidence provided in the manuscript to support this. This statement is purely speculative. The authors should avoid making these types of statements without having any supportive data.

Line 362-368: All the above-mentioned issues must be addressed before making these i-iv conclusions. In it’s current form, the provided is not sufficient to make these conclusions.

Line 369-370: NO. This statement is totally speculative and should be rephrased.

Reviewer #2: Comments:

Abstract:

Its better to mention monocyte-derived macrophages (MDMs) in the abstract (line: 14, 21, 22, and rest of the manuscript). Because authors used MDMs rather than macrophages isolated from lung lavage and/or peritoneal cavity for the phagocytosis assay. It would be more informative to mention authors measured TNF-alpha by ELISA (abstract and other places) rather than just typing TNF as it has been mentioned in Tette et al., 2015 publication.

Materials and Methods:

Line 74: Define PBS

Line 75: change , or CFU/mL to (CFU/mL),

Line 76: define PPLO

Line 78: start the sentence with Twenty uL (not 20uL), were the tubes prepared in triplicate and/or spotted three 20uL on the plate?

Line 83: this author is not familiar with the term “non-decomplemented bovine sera” but rather familiar with “non-heat-inactivated sera”.

Line 88: remove at 19-23C

Line 93-94: change “The specific Mmm antiserum” to “The Mmm specific antiserum”

Line 97: this authors usually writes “complement was inactivated by heating….” rather than “decomplemented ….”

Line 99: change Macrophages in the title to “Monocyte-derived macrophages”

Line 109: define MOI and also typically MOI is written as 1:1; 1:10; or 1:100. This author typically write as below “multiplicity of infection (MOI) at a 1:10 ratio (monocytes: bacteria)”

Line 111: did the authors use any other percentage of sera for the assays such as 50% v/v and 90% v/v etc.

Line 114: why did not authors use Trypsin/EDTA or cold EDTA to lift the cells?

Line 127: what is the final concentration of PI? (6 ug/mL?)

Line 135: authors could define excitation and emission wave lengths for GFP and PI and the band width of filters used.

Line 146: TNF-alpha?

Line 152-155: its better to describe here how the data were presented such as mean and standard deviations (SD) [of CFU or optical densities (OD)] etc.

Results:

Line 165: is direct cytotoxicity of complement on Mmm due to the formation of membrane attack complex through alternative and/or C-type lectin activation complement pathway?

Line 166: since serum heat inactivation condition is described under methods (line 97), 56C, 30 can be removed and modify the sentence accordingly. Move the reference to line 97.

Line 176 and in other figures, just indicate * and P value since authors have already defined significance under the statistics section (line 154-155).

Line 182-184: in Fig. 1a, authors have clearly demonstrated significant killing activity with 10% serum samples from two of the three animals during 1 hr incubation. It is not clear to this reviewer then why the 10% serum had no effect when in the Fig. 2. Also why did authors used 5-10% serum vs 20% Mmm-specific antiserum knowing that 20% non-decomplemented serum can kill Mmm unless antisera were heat-inactivated (but this reviewer could not find this information from the method).

Line 189: are you suggesting here killing of Mmm with specific antisera are due to the activation of classical compliment pathway (and/or Fc-receptor mediated phagocytosis)?

Line 205-206: it was a good idea to use M. bovis as a control for gentamicin protection/killing assay.

Line 217-239: was the Mmm-specific antisera heat inactivated? If so, this phagocytosis most likely through Fc-receptor pathway.

Line 273-282: it’s better to mention authors measured TNF-alpha here because mAb used were TNF-alpha specific (Totte et a., 2015 study)

Line 273-282: the lack of TNF-alpha at lower MOI is intriguing since Sacchini et al., 2012 indicated the presence of several cytokines including TNF-alpha when cattle were challenged with Mmm. Does this mean TNF-alpha is produced by other type of cells under lower MOI (such as B-cells, NK cells, endothelia cells, fibroblast etc.?

Discussion:

Overall, nicely written discussion.

In general, the lack of phagocytosis in the presence of serum compliment is an interesting observation. Macrophages typically have all the complement receptors such as CR1, CR3, CD4 etc for efficient complement-mediated phagocytosis. Unlike findings in this study, phagocytosis of other Mycoplasma species is CR-dependent. Mmm-specific serum mediated phagocytosis and the lack of phagocytosis in the presence of cytochalasin-D is also interesting and informative finding. It would have been ideal if authors used heat-inactivated antiserum to clearly indicate involvement of FC-receptors. Additionally, authors could have also adsorbed Mmm-specific Abs in the antisera and then perform the phagocytosis assays to definitively confirm the role of Ab. Similar studies have been done for M. bovis (Prysliak et al., 2023). The suggestion of C-type lectin receptor mediated entry into macrophages warrant additional studies. As authors have described in line 349, it is intriguing why the MIB-MIP system failed to cleave antibodies. If the whole genome sequencing data is available to Mmm 8740-Rita strain, authors could check for the presence (or absence) of MIB-MIP genes.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Rohana P. Dassanayake

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Comments to authors_PONE-D-23-32412.docx

pone.0305851.s003.docx^{(16KB, docx)}

PLoS One. 2024 Jun 27;19(6):e0305851. doi: 10.1371/journal.pone.0305851.r002

Author response to Decision Letter 0

19 Feb 2024

Answers to reviewer #1:

1/ Line 108-109: In this manuscript, the methodology used to study the interaction of Mmm and macrophages is extremely important. So, Mmms were left with the macrophages for 1 hr in all the experiments?

Yes.

2/Were the cells washed before adding different serums.

Yes since the medium was replaced as stated line 107.

3/The way methodology is written currently, it seems that both mycoplasmas and serums were added together. It is very important for the results observed in Fig 2 because if the mycoplasmas and serums were added together then its really not different compared to the data presented in Fig 1.

The authors need to clarify this.

Experiments in Fig1 are performed in the absence of macrophages whereas in Fig 2 mycoplasmas and serums are added to macrophages. In addition, bovine sera were used at non-bactericidal concentrations in Fig2’s experiment. Incubation of mycoplasmas with bovine sera for 30min before addition to macrophages was also tested and gave similar results (see lines 356-358 in first version).

4/ Flow cytometry analysis (Line 130-132): The flow cytometry analyses are important in order to understand whether Mmms are internalised (gone inside of the macrophages) or associated (made complex and stuck outside) with macrophages. Based-on the current gating strategy how would you differentiate macrophage-associated Mmm from macrophage-internalised Mmm?

By the use of propidium iodide (PI) as stated lines 144-145. See also answer to question 4/ below.

5/ Given that Mmms are so small, it will be very challenging to differentiate the macrophage-associated Mmm from macrophage-internalised Mmm. Due to this limitation, both the externally bound Mmm (large proportion) and internalised (minimum proportion) to macrophages will generate the same double positive signal (FITC + PI).

As stated in sub-chapter “flow cytometry” of chapter “Mat&Met”, propidium iodide (PI) is added after harvesting macrophages after 1h incubation with mycoplasmas, and at 4°C, and for 10min only. In other words, and since PI does not enter viable macrophages, the only situation where internalized Mmms would also be positive for PI is if they are taken up by macrophages during that 10min incubation time at 4°C which prevents phagocytosis. In contrast, the majority of mycoplasmas internalized during the 1h incubation at 37°C in the absence of PI cannot be positive for PI.

6/ Line 180: Based on the methodology, it doesn't seem that the authors have focused on Mmm associated to the macrophages. Please clarify.

In flow cytometry, a size-based gate is commonly used to exclude cell-debris. Such a gate will also contain mycoplasmas due to their small size. As explained above, larger mycoplasmas aggregates will be positive for PI and excluded from the analysis.

7/Line 181: The authors mention that "no cytopathological effects were detected on macrophages". Did the authors measure viability of the macrophages?

No, only adherence and morphological features were observed under a light microscope. Since macrophages adhere to and spread on plastic, any cytopathological effect would result in cell rounding and/or loss of adhesion.

8/Line 186: This is bit confusing. MOI is always represented in relation to cells, but the authors are saying in the absence of macrophages. Please clarify.

A supplementary figure (S2 Fig) was added to show results obtained in the absence of macrophages.

9/Line 205: How was this conclusion made? The authors just mentioned that that "overwhelming majority of Mmm survival at 24 h was due to extracellular mycoplasmas". I don't think that the authors can make this conclusion based on the current results. First, it needs to be demonstrated that Mmm can survive in macrophages. Given that very low number of Mmm were detected in the G+ treatment group, it is almost impossible to say that "complement had no effect on Mmm survival inside macrophages".

The gentamicin assay is a classical assay used since many years to address the question of intracellular survival of mycoplasmas. Only intracellular bacteria will survive in the presence of gentamicin in the medium. Here we show that CFU titers in mixed Mmm+macrophages cultures dramatically drops when gentamicin is added to the medium. Thus, indicating that the vast majority of Mmm survival at 24 h was due to extracellular mycoplasmas. If complement had a significant effect on intracellular survival of Mmm, it would substantially increase the CFU titer which is not the case.

10/ Line 220: These results are contradictory to the previous observations presented in Fig 3. In that, hardly any Mms were internalised but here the authors claim that over 90% were engulfed by the macrophages. Please clarify these contradicting results.

Fig 3 does not give information on the amount of Mmm being internalized but on Mmm survival inside macrophages. In Fig 4, fluorescence of macrophages due to internalization of neongreenMmm was observed but only at high MOIs (i.e., MOI>1:100, which is 10 times the MOI used in Fig3’s experiment). Unfortunately, intracellular survival could not be assessed at that MOI since gentamicin was not 100% bactericidal. This has been addressed in the discussion lines 334-337 of the old version of the manuscript: “Unfortunately, gentamicin was not 100% bactericidal at MOIs used for flow cytometry, thus preventing assessment of the viability of intracellular Mmm. The lack of decrease in fluorescence over time suggests that either no killing occurred or that it was not sufficient to cope with ongoing growth of Mmm on macrophages.”.

11/ Line 222: While it is understandable the rational of using neogreen expressing strain, I think there is bigger fundamental problem here. Given that no FITC signal was detected when MOI of 100 or less were used, it clearly indicates that both the WT and neogreen expressing strains are not behaving the same when it comes to their ability to infect or engulfed by macrophages. The first question, which must be answered that how comparable these two different strains are in terms of their ability to internalise or engulfed by macrophages. As far as I see, there is no data on this. Therefore, no conclusion can be made from any comparisons of the mutant and WT Mmm strains in this study.

The WT Mmm are used to obtained the background level of fluorescence. The lack of fluorescence above that background obtained with neongreen Mmm at an MOI of 100 does not exclude engulfment by macrophages. It can simply be a matter of sensitivity of the assay due to high background fluorescence of WT Mmm. Nevertheless, at higher MOIs, neongreen Mmm gave up to one-log increase in fluorescence which is very significant, albeit low, suggesting low numbers of Mmm being engulfed by each macrophage.

12/Line 226: Given that the uptake (internalisation) is low, this conclusion cannot be made.

This has nothing to do with gating but is due to the choice of a logarithmic scale for FITC fluorescence. Lowering the sensitivity setting for FL1/FITC was tested and indeed allowed calibrating the peak signal given by WT Mmm around zero. However, this had the effect of compressing the signals and was masking the slight increase in fluorescence produced by neongreen Mmm in the absence of antiserum.

13/ Line 275: Mmm were diluted in PBS. Can the authors provide the source of PBS and other reagents used in the cell-culture experiments? Given that TNF levels can be easily induced even by a small amount of co-contaminating endotoxin levels in reagents, it is absolutely important to use certified endotoxin free reagents in cell culture experiments.

PBS alone was tested in pilot experiment and shown to have no effect on background TNF levels in supernatant from macrophages cultures.

14/Line 278-279: I don't think, that is the correct way of reporting experimental findings. That's the variability of the outbred animals. You can't just take two best readings out of three. The results from the third animal must be reported. Otherwise, this experiment becomes, redundant.

There was no effect of Mmm infection with or without complement on that one animal most likely because the background control (i.e., medium+FCS only) was very high. Therefore, it was removed from the analysis. It is common when using large outbred animals to exclude those that show activation in the absence of stimuli because due to financial constraint small animal numbers are used.

15/Line 292: Fig 5 so, 2 animals each for serum 1 and serum 2? Is that correct?

Yes that is correct.

16/Line 315: Yes, this is a most likely scenario, but authors kept saying that Mmms have been internalised. So, results need to be updated.

No, because there can be internalization without intracellular survival

17/Line 319: The authors should avoid using "internalisation or intracellular" because based on the results presented these conclusions can't be made. The current flow cytometry analyses are not sufficient to conclude that Mmms are internalised into macrophages. Other methodology such as confocal microscopy could be used to analyse internalisation of Mmm into macrophages.

As explained above, the flow cytometry method used in our study is tailored to target intracellular Mmm and has been validated previously by our group using confocal microscopy

(DOI: 10.1016/j.jbiotec.2016.08.006)

18/Line 345: But the authors just mentioned (in line 339) that the uptake of Mmm was independent of phagocytosis. Please clarify and discuss these in the discussion section.

This section refers to cultures were anti-Mmm antiserum (specific opsonization) has been added whereas line 339 refers to the presence of bovine complement (non-specific opsonization).

19/Line 346: Again, the intensity can also increase if Mmm + macrophages form a complex and not necessarily internalised. So, the gating strategy must be presented with side and forward scatter graphs.

See explanation above for exclusion from the analysis of extracellular Mmm bound to macrophages membranes.

20/Line 352: Why did the authors not comment on the third animal, which showed substantial increase in TNF levels. The impact of that on the study should be discussed here.

That animal was removed from results due to high background responses (see point 14).

Answers to reviewer #2:

Abstract:

1/Its better to mention monocyte-derived macrophages (MDMs) in the abstract (line: 14, 21, 22, and rest of the manuscript). Because authors used MDMs rather than macrophages isolated from lung lavage and/or peritoneal cavity for the phagocytosis assay.

Macrophages has been replaced by MDMs throughout the manuscript.

2/It would be more informative to mention authors measured TNF-alpha by ELISA (abstract and other places) rather than just typing TNF as it has been mentioned in Tette et al., 2015 publication.

TNF has been replaced by TNF-alpha throughout the manuscript.

Materials and Methods:

1/Line 74: Define PBS. Done.

2/Line 75: change , or CFU/mL to (CFU/mL), Done.

3/Line 76: define PPLO. Done.

4/Line 78: start the sentence with Twenty uL (not 20uL), were the tubes prepared in triplicate and/or spotted three 20uL on the plate? Done. Experiments were performed in triplicates not titration.

5/Line 83: this author is not familiar with the term “non-decomplemented bovine sera” but rather familiar with “non-heat-inactivated sera”. “Non-heat-inactivated” was added line 93.

6/Line 88: remove at 19-23C. Done.

7/Line 93-94: change “The specific Mmm antiserum” to “The Mmm specific antiserum”. Done.

8/Line 97: this author usually writes “complement was inactivated by heating….” rather than “decomplemented ….”.

The sentence was changed accordingly in lines 109-110.

9/Line 99: change Macrophages in the title to “Monocyte-derived macrophages”. Done.

10/Line 109: define MOI and also typically MOI is written as 1:1; 1:10; or 1:100. This author typically write as below “multiplicity of infection (MOI) at a 1:10 ratio (monocytes: bacteria)”.

The sentence was changed lines 124-125 and elsewhere in the manuscript.

11/Line 111: did the authors use any other percentage of sera for the assays such as 50% v/v and 90% v/v etc.

No since the objective of the study was to use sera at non-bactericidal concentrations.

12/Line 114: why did not authors use Trypsin/EDTA or cold EDTA to lift the cells?

That would require an additional step to wash the cells before adding Trypsin in order to remove trace amount of FCS that is present in the medium.

13/Line 127: what is the final concentration of PI? (6 ug/mL?)

100 ug/mL.

15/Line 135: authors could define excitation and emission wave lengths for GFP and PI and the band width of filters used.

This information can easily be found on the internet or on the manufacturer’s websites.

16/Line 146: TNF-alpha? Done.

17/Line 152-155: its better to describe here how the data were presented such as mean and standard deviations (SD) [of CFU or optical densities (OD)] etc.

This information is already provided in each sub-chapter of the materials and methods chapter.

Results:

1/Line 165: is direct cytotoxicity of complement on Mmm due to the formation of membrane attack complex through alternative and/or C-type lectin activation complement pathway?

2/Line 166: since serum heat inactivation condition is described under methods (line 97), 56C, 30 can be removed and modify the sentence accordingly. Move the reference to line 97.

Done.

3/Line 176 and in other figures, just indicate * and P value since authors have already defined significance under the statistics section (line 154-155).

Done.

4/Line 182-184: in Fig. 1a, authors have clearly demonstrated significant killing activity with 10% serum samples from two of the three animals during 1 hr incubation. It is not clear to this reviewer then why the 10% serum had no effect when in the Fig. 2. Also why did authors used 5-10% serum vs 20% Mmm-specific antiserum knowing that 20% non-decomplemented serum can kill Mmm unless antisera were heat-inactivated (but this reviewer could not find this information from the method).

Serum from these animals were used at 5% in Fig2’s experiment. Mmm-specific antiserum was heat inactivated prior to use and as mentioned line 97.

5/Line 189: are you suggesting here killing of Mmm with specific antisera are due to the activation of classical compliment pathway (and/or Fc-receptor mediated phagocytosis)?

Fc-receptor mediated phagocytosis since Mmm-specific antiserum is heat-inactivated.

6/Line 217-239: was the Mmm-specific antisera heat inactivated? If so, this phagocytosis most likely through Fc-receptor pathway.

Yes.

7/Line 273-282: it’s better to mention authors measured TNF-alpha here because mAb used were TNF-alpha specific (Totte et a., 2015 study)

Done.

8/Line 273-282: the lack of TNF-alpha at lower MOI is intriguing since Sacchini et al., 2012 indicated the presence of several cytokines including TNF-alpha when cattle were challenged with Mmm. Does this mean TNF-alpha is produced by other type of cells under lower MOI (such as B-cells, NK cells, endothelial cells, fibroblast etc.?

It is difficult to know precisely what are the MOIs in vivo in infected alveolar spaces. Mmm concentrations in pleural fluid of diseased animals were reported to reach up to 1012 CFU/mL whereas, in this study, 1.5 x 107 to 1.5x108 CFU/mL were used to observe TNF-alpha production by infected MDMs. It would be very interesting to study interactions of Mmm with other immunocompetent cells.

Discussion:

1/It would have been ideal if authors used heat-inactivated antiserum to clearly indicate involvement of FC-receptors.

This was the case in our study as explained above in points 4-6 of “results”.

2/Additionally, authors could have also adsorbed Mmm-specific Abs in the antisera and then perform the phagocytosis assays to definitively confirm the role of Ab. Similar studies have been done fo

PLoS One. doi: 10.1371/journal.pone.0305851.r003

Decision Letter 1

Rohana P Dassanayake

2 Apr 2024

PONE-D-23-32412R1

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions

PLOS ONE

Dear Dr. Totté,

Please submit your revised manuscript by May 17 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Rohana P Dassanayake

Guest Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

________________________________________

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3:

Reviewer #4: All comments have been addressed

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

6. Review Comments to the Author

Reviewer #3: Comments to authors

The article investigated the interactions between Mycoplasma mycoides subsp. mycoides and bovine monocyte-derived macrophages, in presence or absence of different concentrations of complement or anti-Mmm antiserum.

The research provides a relevant contribution to further understanding the mechanisms of host-pathogen interaction that occur in the early stages of contagious bovine pleuropneumonia (CBPP). Macrophages (namely alveolar macrophages) represent the first line of defence in the lung where the infection takes place. The results demonstrated that Mmm specific antibodies enhance phagocytic and bactericidal activity of MDMs. Importantly these data confirm that high concentrations of Mmm are able to directly induce macrophages to produce TNF-alpha, a pro-inflammatory cytokines that correlate with severe CBPP disease.

In general the article is well written and the methodology applied was functional to answer the research questions investigated.

Results are clearly presented.

Minor comments.

Conclusions

Considering the very interesting work done, I would recommend the authors to integrate the discussion commenting the results within the context of CBPP pathogenesis to increase the significance and the importance of the research. Find below some aspect to consider:

Use of MDMs. One of the main challenge in CBPP research is the difficulty of investigating the host-pathogen interaction at lung level in the early stages of disease onset. Due to the lack of laboratory animal models of CBPP infection, we urgently need simplified in vitro and ex vivo models to dissect the complex cattle immune response leading to disease pathology. Even if the ideal target cells to be investigated are the alveolar macrophages, harvesting of these cells from in vivo is not straightforward and require expert animal manipulation. The use of MDMs represent a valid alternative and it provided similar results with the advantage of starting from a simple blood sampling.

Complement. What is the current knowledge on the role of complement in the lung? May the complement contribute to down modulate the macrophage activation during the primary phase of infection and mycoplasma replication? Or the abrogation of TNF-alpha production by MDMs observed in presence of bactericidal concentrations of complement is simply related to the reduction of Mmm concentration?

TNF-alpha. High concentrations of Mmm can induce TNF-alpha release by macrophages even in the absence of anti-Mmm antiserum. May this explain why there is no correlation between severe pathological findings and antibody response in CBPP infected animals? What is the role of these cytokine in cbpp pathogenensis and lung pneumonia? More importantly, what is the immune networking that is activated by tnf-alpha? Can the induction of TNF-alpha initiate the immune mechanisms that lead to CBPP fibrinous pneumonia?

Furthermore only high concentration of Mmm induced production of TNF-alpha. This indicates that while Mmm is replicating at low concentrations the immune system is not activated. May this explain why, in natural conditions, naïve animals requires multiple or prolonged exposure to diseased animal to get infected? Similarly, may this support the long incubation time sometimes observed in infected animals?

Other comments

Minor editing corrections are required in the paragraph results Effect of bovine complement on the uptake of fluorescent Mmm by macrophages: line 232-257 (some errors: of of, MIF, FIg4c)

________________________________________

Reviewer #4: Comments to authors:

Based on my review, I find the manuscript acceptable for publication. The authors have responded to the previous reviewer's comments well.

The knowledge generated from this study is crucial in enhancing our understanding of the Mmm interaction with macrophages.

Abstract:

Line 202: Write the abbreviation “MOI” in full

Introduction:

Line 36: Write WOAH in capital letters

Results:

Line 181: the sentence is not clear. It may be replaced with … “The mycoplasmacidal activity and inter-animal variability of bovine sera remained consistent throughout the study”.

Line 215: The efficacy of antibiotics against mycoplasmas is restricted to specific mycoplasma species and only reduces the concentration of mycoplasmas rather than killing the bacteria. Why was gentamycin used when there are more effective antibiotics like Tyrosin?

Discussion:

Line 336: It’s not clear. This information indicates that gentamicin was effective in preventing the survival of the bacteria within the macrophages, leading to the conclusion that the majority of the viable bacteria were located on the cell surfaces. Why was the gentamycin effective in this case?

Line 376: Use a uniform method of citation.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: Yes

________________________________________

Do you want to get recognition for this review on a Web of Science researcher profile?

If you opt in, your Web of Science profile will automatically be updated to show a verified record of this review in full compliance with the journal’s review policy. If you don’t have a Web of Science profile, you will be prompted to create a free account.

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

Attachment

Submitted filename: Manuscript Number PONE-D-23-32412 reviewers comments.docx

pone.0305851.s004.docx^{(13KB, docx)}

PLoS One. 2024 Jun 27;19(6):e0305851. doi: 10.1371/journal.pone.0305851.r004

Author response to Decision Letter 1

4 Jun 2024

see response to reviewers

Attachment

Submitted filename: ResponseToReviewersPONE-D-23-32412R2.docx

pone.0305851.s005.docx^{(16.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0305851.r005

Decision Letter 2

Rohana P Dassanayake

6 Jun 2024

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions

PONE-D-23-32412R2

Dear Dr. Totte,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Rohana P Dassanayake

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0305851.r006

Acceptance letter

Rohana P Dassanayake

18 Jun 2024

PONE-D-23-32412R2

PLOS ONE

Dear Dr. Totté,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Rohana P Dassanayake

Guest Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Stability of the bactericidal effect of bovine complement.

(TIF)

pone.0305851.s001.tif^{(119.9KB, tif)}

S2 Fig. Survival of Mmm in vitro in supplemented IMDM medium in the absence of macrophages.

(TIF)

pone.0305851.s002.tif^{(95.7KB, tif)}

Attachment

Submitted filename: Comments to authors_PONE-D-23-32412.docx

pone.0305851.s003.docx^{(16KB, docx)}

Attachment

Submitted filename: Manuscript Number PONE-D-23-32412 reviewers comments.docx

pone.0305851.s004.docx^{(13KB, docx)}

Attachment

Submitted filename: ResponseToReviewersPONE-D-23-32412R2.docx

pone.0305851.s005.docx^{(16.5KB, docx)}

Data Availability Statement

Data are available online at https://zenodo.org/record/7442582#.ZAiYrHbMJOC.

[pone.0305851.ref001] 1.World Organization for animal Health (2015). https://www.woah.org/en/what-we-do/animal-health-and-welfare/animal-diseases/?_alphabet=C. Accessed 20 Sept 2022

[pone.0305851.ref002] 2.Provost A, Perreau P, Bréard A. Contagious bovine pleuropneumonia (1987) Rev Sci Tech Off. int. Epiz., 6 (3), 625–679. 10.20506/rst.6.3.306 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref003] 3.Scanziani E, Paltrinieri S, Boldini M, Grieco V, Monaci C, Giusti AM, et al. (1997) Histological and immunohistochemical findings in thoracic lymph nodes of cattle with contagious bovine pleuropneumonia. J Comp Pathol. Aug;117(2):127–36. doi: 10.1016/s0021-9975(97)80029-1 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref004] 4.Jungi TW, Krampe M, Sileghem M, Griot C, Nicolet J (1996) Differential and strain-specific triggering of bovine alveolar macrophage effector functions by mycoplasmas. Microb Pathog. 2:487–498. doi: 10.1006/mpat.1996.0078 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref005] 5.Volanakis J.E. (1998) Overview of the complement system, in: Volanakis J.E., Frank M.M., (Eds.), The human complement system in health and disease, Marcel Dekker Inc., New York, 1998, pp. 9–32. doi: 10.1201/9780367800772 [DOI] [Google Scholar]

[pone.0305851.ref006] 6.Cosma CL, Sherman DR, Ramakrishnan L. (2003) The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol 57:641–76. doi: 10.1146/annurev.micro.57.030502.091033 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref007] 7.Horwitz M. A. (1983) The Legion’aires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108–2126. 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0305851.ref008] 8.Okusawa S, Yancey KB, van der Meer JW, Endres S, Lonnemann G, Hefter K, et al. (1988) C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha. J Exp Med. 1;168(1):443–8. doi: 10.1084/jem.168.1.443 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0305851.ref009] 9.Laudisi F, Spreafico R, Evrard M, Hughes TR, Mandriani B, Kandasamy M, et al. (2013) Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1β release. J.Immunol. Aug 1;191(3):1006–10. 10.4049/jimmunol.1300489 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0305851.ref010] 10.Marshall AJ, Miles RJ, Richards L. (1995) The phagocytosis of mycoplasmas. J Med Microbiol. Oct;43(4):239–50. doi: 10.1099/00222615-43-4-239 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref011] 11.Bonnefois T, Vernerey MS, Rodrigues V, Totté P, Puech C, Ripoll C, et al. (2016) Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades. J Biotechnol. Oct 20;236:35–44. doi: 10.1016/j.jbiotec.2016.08.006 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref012] 12.Yaya A, Golsia R, Hamadou B, Amaro A, Thiaucourt F. Essai comparatif d’efficacité des deux souches vaccinales T1/44 et T1sr contre la péripneumonie contagieuse bovin. Rev Elev Med Vet Pays Trop. 1999;52(3–4):171–179. https://www.vetres.org/articles/vetres/pdf/2006/05/v6049.pdf [Google Scholar]

[pone.0305851.ref013] 13.Le Goff C., Thiaucourt F., 1998. A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP). Vet. Microbiol., 60: 179–191. https://www.sciencedirect.com/science/article/pii/S0378113598001564 doi: 10.1016/s0378-1135(98)00156-4 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref014] 14.Suleman M, Prysliak T, Clarke K, Burrage P, Windeyer C, Perez-Casal J. (2016) Mycoplasma bovis isolates recovered from cattle and bison (Bison bison) show differential in vitro effects on PBMC proliferation, alveolar macrophage apoptosis and invasion of epithelial and immune cells. Vet Microbiol. Apr 15;186:28–36. 10.1016/j.vetmic.2016.02.016 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref015] 15.Totté P, Puech C, Rodrigues V, Bertin C, Manso-Silvan L, Thiaucourt F. (2015) Free exopolysaccharide from Mycoplasma mycoides subsp. mycoides possesses anti-inflammatory properties. Vet Res. Oct 21;46:122. doi: 10.1186/s13567-015-0252-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0305851.ref016] 16.Gaurivaud P, Lakhdar L, Le Grand D, Poumarat F, Tardy F. (2014) Comparison of in vivo and in vitro properties of capsulated and noncapsulated variants of Mycoplasma mycoides subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine pleuropneumonia. FEMS Microbiol Lett. Oct;359(1):42–9. 10.1111/1574-6968 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref017] 17.Schieck E, Lartigue C, Frey J, Vozza N, Hegermann J, Miller RA, et al. (2016) Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance. Mol Microbiol. Jan;99(1):55–70. 10.1111/mmi.13213. [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref018] 18.Howard CJ, Taylor G, Collins J, Gourlay RN. (1976) Interaction of Mycoplasma dispar and Mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leukocytes. Infect Immun. Jul;14(1):11–7. doi: 10.1128/iai.14.1.11-17.1976 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0305851.ref019] 19.Buttery S.H. (1970) Hapten inhibition of the reaction between Mycoplasma mycoides polysaccharide and bovine antisera. Immunochemistry, 7, 305–310. doi: 10.1016/0019-2791(70)90168-0 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref020] 20.Weis J.J., Law S.K., Levine R.P., Cleary P.P., (1985) Resistance to phagocytosis by group A streptococci: Failure of deposited complement opsonins to interact with cellular receptors, J. Immunol. 134 (1985) 500–505. [PubMed] [Google Scholar]

[pone.0305851.ref021] 21.Lindenwald DL, Monteiro JT, Rautenschlein S, Meens J, Jung K, Becker SC, et al. (2020) Ovine C-type lectin receptor hFc-fusion protein li–rary—A novel platform to screen for host-pathogen interactions. Vet Immunol Immunopathol. Apr 18;224–230. 10.1016/j.vetimm.2020.110047 [DOI] [PubMed] [Google Scholar]

[pone.0305851.ref022] 22.Arfi Y, Minder L, Di Primo C, Le Roy A, Ebel C, Coquet L, et al. (2016) MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G. Proc Natl Acad Sci U S A. May 10;113(19):5406–11. doi: 10.1073/pnas.1600546113 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions

Philippe Totté

Tiffany Bonnefois

Lucia Manso-Silván

Roles

Abstract

Introduction

Methods

Bacterial strains

Bovine complement and antiserum

Macrophages and bacterial infection

Flow cytometry

ELISA

Statistics

Results

Bactericidal effect of bovine complement on Mmm viability in the absence of macrophages

Fig 1.

Effect of bovine complement on Mmm opsonization and survival in the presence of macrophages

Fig 2. Macrophages do not kill Mmm even in the presence of bovine complement.

Fig 3. Bovine complement has no impact on Mmm survival inside macrophages.

Effect of bovine complement on the uptake of fluorescent Mmm by macrophages

Fig 4. Bovine complement does not impact Mmm uptake by macrophages.

Table 1. Effect of bovine complement (serum) from three different animals (mean+/-SD) on the uptake of fluorescent Mmm by autologous macrophages as measured by flow cytometry 1 h and 24 h post infection.

Effect of bovine complement on Mmm-induced TNF production by macrophages

Fig 5. Effect of bovine complement on Mmm-induced release of TNF by autologous macrophages.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Jianhong Zhou

Roles

Author response to Decision Letter 0

Decision Letter 1

Rohana P Dassanayake

Roles

Author response to Decision Letter 1

Decision Letter 2

Rohana P Dassanayake

Roles

Acceptance letter

Rohana P Dassanayake

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases