Key molecules of Mucorales for COVID-19-associated mucormycosis: a narrative review : Journal of Bio-X Research

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Key molecules of Mucorales for COVID-19-associated mucormycosis: a narrative review

Baberwal, Priyanka; Singh, Arjun; Adarsh, Abhinav; Kumar, Yatender*,

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doi: 10.1097/JBR.0000000000000131
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Abstract

Introduction

101 mucormycosis cases in patients with coronavirus disease 2019 (COVID-19) were documented by May 13, 2021, with 82 cases in India and 19 in the rest of the world.[1] Pre-existing diabetes mellitus was found in 80% patients; in 14.9% cases, diabetic ketoacidosis (DKA) was present.[1] Corticosteroids were taken for COVID-19 therapy in 76.3% patients. Nasal and sinus mucormycosis was the most common (88.9%), followed by rhino-orbital mucormycosis (56.7%).[1] Among the total cases reported for mucormycosis, the fatality rate was 30.7%.[1]

Invasive mucormycosis is divided majorly into six clinical forms based on its clinical manifestations and anatomic location: rhinocerebral infection, pulmonary infection, cutaneous infection, gastrointestinal infection, disseminated infection, and other unusual, rare types such as endocarditis and renal infection.[2,3] People typically develop this infection via inhalation or ingestion of Mucorales mould spores, or also by other percutaneous routes. Mucormycosis frequently causes infarction of the affected tissues, thrombosis, and tissue damage owing to a range of fungal proteases, lipases, and mycotoxins. Disseminated mucormycosis is frequently caused by a delayed diagnosis of this disease.[4]

The significant mortality rate associated with this condition is due to a lack of adequate treatment and prompt detection. Several studies on Mucorales genes, pathways, and mechanisms have been published, demonstrating a direct link between virulence and prospective therapeutic and diagnostic targets.[5,6] Several proteins such as high-affinity iron permease (FTR1),[7] spore coat protein (CotH),[8] calcineurin (CaN),[9] and ADP-ribosylation factors (ARFs)[10] are involved in the pathogenesis. This review discusses the potential of these molecules to be prospective targets for the development of new diagnostic and therapeutic methods for mucormycosis.

Database search strategy

For relevant species that cause mucormycosis, searches were made in the Uniprot and NCBI Genbank databases. The sequences of following species which cause mucormycosis were retrieved – Rhizopus microspores, Rhizopus Azygosporus, Rhizopus Delemar, Rhizopus Oryzae, Mucor Circinelloides, Mucor lusitanicus, Syncephalastrum racemosum, Lichtheimia corymbifera, Rhizopus stolonifera, Mucor Ambiguus from the databases on basis of the keywords of the protein FTR1 (A0A1X0RSJ3, A0A367JU44, I1BRD6, A1JHN4, A1JHP3, A0A168IGR0, A0A1X2HRM4, A0A068RGG6, A0A367IPH9, A0A0C9N157), ARF (A0A1X0S2D7, A0A367KAL2, KAG1467656.1, KAG1108544.1, S2JQ90, A0A168M996, A0A1X2HFW9, A0A068SD20, A0A367KFG1, A0A0C9MMV9), CaN (A0A2G4SQR0, A0A367IYH0, I1CCM5, KAG1185535.1, S2J3F1, A0A168LIB8, A0A1X2HHC6, A0A068S9K6, A0A367KPU7, A0A0C9MJ19) and CotH (A0A2G4SYA1, A0A367JFE6, I1BJ20, KAG0767446.1, S2JW22, KAF1802881.1, A0A1X2HTS9, A0A068RYS2, A0A367J2C2, A0A0C9MP78).

FTR1

FTR1 is a high-affinity iron permease protein, which is necessary for iron uptake in iron-depleted environments. At the molecular level, FTR1 has high-affinity ferrous iron transmembrane transporter activity in fungi. It also plays a major biological role in the pathogenesis of mucormycosis and reductive iron assimilation.[11] High-affinity iron permease is a component of a reductive system that includes redundant surface reductases that convert ferric to ferrous, which is more soluble. The reduced ferrous iron generated by the surface reductase is captured by a protein complex comprising a multicopper oxidase and a ferrous permease.[12]

In patients suffering from DKA, the production of free iron ions increases in the blood, causing impairment of iron homeostasis[13] COVID-19 also interacts with the hemoglobin molecule, causing iron to dissociate from hem molecules, resulting in hyperferritinemia in COVID-19 patients.[14] Hyperferritinemia is caused by both hyperglycemia and COVID-19, and excessive intracellular ferritin induces the release of oxygen-free radicals, which destroys the tissue and releases free iron into the circulation. Mucorales development and invasion of blood arteries is aided by free iron in the blood; this is facilitated by high-affinity iron permease resulting in vascular thrombosis and tissue necrosis.[15,16] Studies on R. oryzae pathogenicity in mucormycosis showed that iron is one of the important nutrients, ie, iron plays an important role as a coenzyme.[17,18]

The high-affinity iron permease (FTR1) has been shown to have a role in mucormycosis pathogenesis in R. arrhizus and Lichtheimia corymbifera. They are also necessary for Mucorales’ development and survival in iron-depleted conditions. Increased expression of FTR1 genes in R. arrhizus was discovered in a study on mice with DKA compared to controls. R. arrhizus can acquire iron in reduced form when the FTR1 gene is disrupted (due to a drop in copy number) or when gene expression is inhibited by RNA interference, resulting in lower pathogenicity in DKA mice.[19] The DKA mice were protected from mucormycosis by using antibodies against the FTR1 gene.[12]

The domain(s) of the FTR1 protein sequence of species causing mucormycosis was analyzed using the Pfam webserver as depicted in Table 1. Proteins with the same domains are more likely to have similar structures and are likely to perform the same cellular function.[20]

Table 1 - FTR1-conserved domains
Name of species Domain Start End Gathering threshold Score (bits) E-value
Sequence Domain Sequence Domain Sequence Domain
Rhizopus microsporus FTR1 1 221 25.30 25.30 159.30 159.00 4E-43 4.9E-43
Rhizopus azygosporus FTR1 7 318 25.30 25.30 237.10 236.80 8.2E-67 1E-66
Rhizopus delemar FTR1 7 314 25.30 25.30 257.10 256.80 6.9E-73 8.3E-73
Rhizopus oryzae FTR1 1 182 25.30 25.30 142.60 142.40 1.8E-37 2.1E-37
Mucor circinelloides FTR1 1 182 25.30 25.30 142.30 142.20 2.3E-37 2.4E-37
Mucor lusitanicus FTR1 7 314 25.30 25.30 236.00 235.70 1.8E-66 2.2E-66
Syncephalastrum racemosum FTR1 7 314 25.30 25.30 246.90 246.60 9E-70 1.1E-69
Lichtheimia corymbifera FTR1 7 313 25.30 25.30 143.10 142.70 3.6E-38 4.6E-38
Note: Pfam was used to identify the conserved domains of the FTR1 protein of different species responsible for causing mucormycosis. FTR1=High-affinity iron permease. Unpublished data.

The consensus sequences of the domains were visualized using Jalview as depicted in Figure 1.[21] Consensus sequences are based on an evolutionary history in which critical residues for protein stability and function have been conserved.[22] Iron plays a fundamental role in the pathogenesis of mucormycosis, and FTR1 helps in the intracellular transfer of iron.[16] Thus, FTR1 may prove to be a key target gene for diagnostics as well as therapeutics in mucormycosis.

F1
Figure 1.:
A comparative analysis of the conserved domain of FTR1 protein sequence of various Mucorales. From the result of Pfam, the conserved domain of FTR1 proteins was analyzed using Jalview. The column marked in blue depicts 90% identity of the residues in the consensus sequence. FTR1=High-affinity iron permease. Unpublished data.

ADP-ribosylation factor

The mucor species can develop into either yeast or hyphal form upon germination, depending on the nutritional and gaseous environment.[23]Mucor circinelloides R7B (leuA) strain was used for the analysis of ARF proteins. ARF1 and ARF2 are 96% identical, and both genes have mutations resulting in different phenotypes.[10] ARFs are localized in the plasma membrane and secretory endosomal and lysosomal pathways.[24] They are involved in protein secretion in yeast.[25] The virulence of ARF mutants was analyzed in mice having diabetes.[26] The ARF1 strain led to 70% death 3 days after inoculation, whereas the ARF2 strain was less virulent and led to 30–40% deaths. The secretory pathway is involved in virulence. Molecules released by M. circinelloides ARF mutant strains show virulence in nematodes. In a mycelial cell-free medium, ARF1 caused the death of nearly half the nematodes, and ARF2 was less virulent.[10] ARF regulates membrane trafficking by a cycle in which nucleotide exchange converts GDP to the GTP-bound form.[27]

The M. circinelloides R7B (leuA) strain was also used for the analysis of ARF-like proteins – ARL1 and ARL2.[28] ARL proteins are localized in trans-Golgi and cytosol microtubules.[29] ARL1 regulates membrane trafficking, and loss of ARL1 alone results in defects in membrane trafficking.[30] ARL2 protein regulates tubulin-folding cofactor D interaction with native tubulin.[31] These proteins are involved in the virulence of mucor species. ARL1’s low expression level during aerobic growth enhances the virulence level. Defects in ARL2 can contribute to a decrease in the mRNA level of ARL1.[28] ARL1 is responsible for the trans-Golgi recruitment of Arfaptin through interaction with their BAR (Bin/Amphiphysin/Rvs) domain-containing region which participates in membrane deformation.[32]

Domains of the ARF and ARL proteins were searched using Pfam as depicted in Table 2. Then, the Jalview visualizer was used to visualize the consensus sequences of the domains as depicted in Figure 2.[21] The protein sequence conservation among all the species of the ARF proteins is highlighted in blue regions.

Table 2 - ARF-conserved domains
Name of species Domain Start End Gathering threshold Score (bits) E-value
Sequence Domain Sequence Domain Sequence Domain
Rhizopus microsporus ARF 6 178 22.3 22.3 260.2 260 2.10E-74 2.40E-74
Rhizopus azygosporus ARF 6 161 22.3 22.3 217.2 216.9 3.60E-61 4.30E-61
Mucor circinelloides ARF 5 177 22.3 22.3 254.5 254.3 1.20E-72 1.40E-72
Mucor lusitanicus ARF 5 177 22.3 22.3 257.4 257.2 1.50E-73 1.80E-73
Syncephalastrum racemosum ARF 5 177 22.3 22.3 257.2 257 1.80E-73 2.00E-73
Lichtheimia corymbifera ARF 5 177 22.3 22.3 258.1 257.9 9.60E-74 1.10E-73
Rhizopus stolonifer ARF 5 177 22.3 22.3 254.8 254.6 9.90E-73 1.10E-72
Mucor ambiguus ARF 5 177 22.3 22.3 257.4 257.2 1.50E-73 1.80E-73
Note: Pfam was used to identify the conserved domains of ARF protein of different species responsible for causing mucormycosis. ARF=ADP-ribosylation factors. Unpublished data.

F2
Figure 2.:
A comparative analysis of the conserved domain of ARL protein sequence of various Mucorales. From the result of Pfam, the conserved domain of ARL proteins was analyzed using Jalview. The column marked in blue depicts 90% identity of the residues in the consensus sequence. ARF=ADP-ribosylation factors. Unpublished data.

The ADP-ribosylation factor (ARF) and ADP-ribosylation factor-like protein (ARL) both are responsible for the virulence in mucor species for mucormycosis in the dimorphism state and proved to be viable targets for diagnostics which might detect the disease during the dimorphism stage.

Calcineurin

CaN is a Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase with two subunits: a catalytic A subunit with phosphatase activity that activates the enzyme complex and a regulatory B subunit that binds to calcium. CaN is involved in dimorphism and virulence among pathogenic fungi.[9] Mucor species are dimorphic fungi and depending on the environment, they can grow as hyphae or yeast. The hyphal form is generally observed in association with diseased tissue.[23]

During the aerobic conditions, Mucor spp. shows hyphal structure, while in anaerobic conditions in the presence of CO2, it exhibits a yeast-like structure.[33] The dimorphism helps fungi in the colonization of the host.[34] CaN is an important component of the dimorphic transition, and this has been proved with the help of the immunosuppressive drug FK506 that inhibits CaN and encourages Mucor to grow exclusively as yeast.[35] Human and fungal CaNs have approximately 80% similarity; whereas, fungal and human FKBP12s (FK506 Binding Protein 12) have a sequence homology of 48–58%.[36] This presents an obstacle for developing anti-fungal drugs against CaN. In both humans and fungi, drug compounds like FK506 and rapamycin bind to the immunophilin FKBP12, and resultant complexes (FK506-FKBP12 and rapamycin-FKBP12) target CaN and TOR. However, owing to their immunosuppressive properties, these medicines cannot be employed as antifungals in their current form. To circumvent this, essential distinctions between human and fungal FKBP12, CaN, and TOR proteins must be identified, allowing for the synthesis of novel small compounds with a higher affinity for fungal components.[37]

The Pfam webserver was used to obtain the domains of the CaN proteins as shown in Table 3. Then, Jalview was used to visualize the consensus sequences of the CaN domains (Fig. 3).[21] The protein sequence conservation among all the species of the CaN proteins is highlighted in blue regions.

Table 3 - Calcineurin-conserved domains
Name of species Domain Start End Gathering threshold Score (bits) E-value
Sequence Domain Sequence Domain Sequence Domain
Rhizopus microsporus Metalo-phosphoesterases 113 315 21.50 21.50 130.40 129.60 4.3E-34 7.4E-34
Rhizopus azygosporus Metalo-phosphoesterases 113 315 21.50 21.50 130.30 129.60 4.3E-34 7.4E-34
Rhizopus delemar Metalo-phosphoesterases 112 305 21.50 21.50 116.10 115.20 9.8E-30 1.8E-29
Mucor circinelloides Metalo-phosphoesterases 105 307 21.50 21.50 130.00 128.60 5.5E-34 1.5E-33
Mucor lusitanicus Metalo-phosphoesterases 99 301 21.50 21.50 129.80 128.60 6.5E-34 1.5E-33
Syncephalastrum racemosum Metalo-phosphoesterases 115 317 21.50 21.50 130.10 129.40 5.1E-34 8.4E-34
Lichtheimia corymbifera Metalo-phosphoesterases 117 319 21.50 21.50 129.60 129.00 7.2E-34 1.2E-33
Serine-threonine protein phosphatase
Rhizopus stolonifer Metalo-phosphoesterases 93 141 26.80 26.80 53.10 51.60 0.00000000011 0.00000000031
Note: Pfam was used to identify the conserved domains of CaN protein of different species responsible for causing mucormycosis. CaN=calcineurin. Unpublished data.

F3
Figure 3.:
A comparative analysis of the conserved domain of CaN protein sequence of various Mucorales. From the result of Pfam, the conserved domain of CaN proteins was analyzed using Jalview. The column marked in blue depicts 90% identity of the residues in the consensus sequence. CaN=calcineurin. Unpublished data.

The CaN pathway is conserved throughout the eukaryotes.[9] Owing to its key role in virulence, CaN may be a potential target for anti-fungal therapeutics and anti-fungal drugs.[38]

CotH

CotH is a protein kinase of the spore coat protein family. It is necessary for the assembly of proteins in the spore coat layer, which is produced during sporulation and is controlled by autophosphorylation with adenosine triphosphate and subsequent phosphorylation.[39] Like spore-forming fungi R. oryzae, it is a necessary component for the development and germination of spores.[8] High glucose levels increase CotH expression on the fungus cell surface and increases free iron availability.[6] This results in GRP78/CotH3 interaction thus, facilitating endothelial/epithelial penetration and fungal spread.[40]

CotH3 is mostly produced during R. oryzae germination and has a stronger ability to attach to and invade the endothelium and nasal epithelial cells. Immunosuppressed mice were protected from mucormycosis by monoclonal antibodies produced against the CotH3 peptide induced by R. delemar and other Mucorales.[8] A study on CotH3 show that it is responsible for the interaction between the pathogen with the cell surface GRP78 of the host.[41]

Domains of the CotH proteins of different species causing mucormycosis were searched using the Pfam web server (Table 4). The consensus sequences of the domains were visualized using Jalview (Fig. 4).[21] The protein sequence conservation among all the species of the CotH proteins is highlighted in the blue regions.

Table 4 - CotH-conserved domains
Name of species Domain Start End Gathering threshold Score (bits) E-value
Sequence Domain Sequence Domain Sequence Domain
Rhizopus microsporus CotH 16 368 23.80 23.80 170.20 169.90 2.8E-46 3.5E-46
Rhizopus azygosporus CotH 194 545 23.80 23.80 168.30 167.60 1E-45 1.7E-45
Rhizopus delemar CotH 176 529 23.80 23.80 161.50 160.80 1.3E-43 2E-43
Mucor circinelloides CotH 215 571 23.80 23.80 158.20 158.20 1.3E-42 1.3E-42
Syncephalastrum racemosum CotH 174 535 23.80 23.80 135.10 131.30 1.3E-35 1.9E-34
Lichtheimia corymbifera CotH 193 515 23.80 23.80 137.30 136.80 2.8E-36 4.1E-36
Rhizopus stolonifer CotH 186 498 23.80 23.80 148.10 147.60 1.4E-39 2.1EE-39
Mucor ambiguus CotH 183 510 23.80 23.80 140.90 140.30 2.3E-37 3.5E-37
Note: Pfam was used to identify the conserved domains of the CotH protein of different species responsible for causing mucormycosis. CotH=spore coat protein. Unpublished data.

F4
Figure 4.:
A comparative analysis of the conserved domain of CotH protein sequence of various Mucorales. From the result of Pfam, the conserved domain of CotH proteins was analyzed using Jalview. The column marked in blue depicts 90% identity of the residues in the consensus sequence. CotH=spore coat protein. Unpublished data.

In Mucorales, the CotH protein helps in the invasion into the host endothelial cells via interacting with the GRP78 of host cells. Experiments have also proven that anti-CotH antibodies have the potential to block the invasion of Mucorales. Hence, targeting the CotH protein of these species might be useful in developing potential diagnostics and therapeutics for mucormycosis.

Phylogenetic analysis

To explore the phylogenetic relationship among the 10 different species (R. microsporus, R. azygosporus, R. delemar, R. oryzae, M. circinelloides, M. lusitanicus, Syncephalastrum racemosum, L. corymbifera, R. stolonifer, and M. ambiguus) of Mucorales for 4 different proteins, namely CaN, ARF, FTR, and CotH, MEGA Version 11 program[42] was used to construct the phylogenetic tree by using the maximum likelihood method.[43] The phylogenetic tree was visualized using iTOL (Fig. 5).[44] The phylogenetic tree is midpoint rooted. Bootstrap values are displayed.

F5
Figure 5.:
Phylogenetic rooted tree based on different protein sequences showing the relationship between different species that are responsible for Mucormycosis. The phylogenetic tree was constructed with the MEGA version 11.0 program by bootstrap analysis using the maximum likelihood method (1000 replicates). Unpublished data.

All Mucorales were divided into four clades with branch length and bootstraps values. All hypothetical proteins lay within the clade of the same proteins. One clade is completely of CaN protein. Lichtheimia corymbifera, R. stolonifer, and S. racemosum are the three outgroups of the CaN protein clade. Mucor ambiguus, M. circinelloides, and M lusitanicus are in one subcluster of the CaN protein clade. The second clade is made up of ARF. The ARF-like protein of R. azygosporus species is present outside the clade of the ARF. The third clade is made of FTR protein. Lichtheimia corymbifera is one outgroup of the FTR clade. Mucor circinelloides and S. racemosum are in the single subcluster of the FTR protein clade. Fourth is the CotH protein and in that clade, M. ambiguus and Lichtheimia are the two outgroups.

Limitations

There are few limitations to this review. Firstly, the number of relevant proteins involved in mucormycosis pathogenesis is relatively small. Secondly, due to the lack of experimental studies, the availability of data for some species was limited. Hence, further research with more data is required and needs to be validated experimentally.

Conclusion

Several Mucorales proteins and their domains were explored in this study to identify key molecules as potential targets for diagnosis as well as therapeutic treatment of CAM. Comorbid conditions in patients with COVID-19, such as iron overload disorder, exert additional risk of being infected by fungus, as FTR1 allows extracellular heme to be taken in and degraded intracellularly by heme oxygenases to produce free iron.[45] Proteins like CaN and ARF are essential for fungi to proliferate. CotH3 and CotH2 facilitate invasion by interacting with host cell GRP78 (which is over-expressed during COVID-19) to mediate pathogenic host-cell interactions.[8,46]

Upon conserved domain analysis for key proteins of different mucormycosis-causing species, ie, FTR1 protein has FTR1 domain, ARF protein has ARF domain, CotH protein has CotH domain, and CaN proteins have metallophosphoesterase domain in all the selected species except R. stolonifer, wherein an additional domain for serine-threonine protein phosphatase N-terminal domain is also present along with the metallophosphoesterase domain in CaN. The study also revealed a hypothetical protein containing CaN domain for R. arrhizus, and an uncharacterized protein containing FTR1 domain for M. circinelloides and R. arrhizus. Phylogenetic tree analysis showed that these four key proteins formed four different clades, and each clade consisted of a particular protein. Future studies focused on these key proteins involved in the pathogenesis might prove to be viable targets for the development of novel mucormycosis diagnostic and therapeutic methods.

Acknowledgments

We sincerely thank the Department of Biological Sciences and Engineering (BSE), Netaji Subhas University of Technology (NSUT), New Delhi, for its technical support and administrative support.

Author contributions

Writing – Original draft preparation: PB, AS, AA; review, editing, and project supervision: YK. All authors approved the final version of the manuscript.

Financial support

None.

Conflicts of interest

There are no conflicts of interest.

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Keywords:

ADP-ribosylation factor; calcineurin; COVID-19-associated mucormycosis; high-affinity iron permease; spore coat protein

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