Variability of the iron uptake system components in Bacillus anthracis

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Introduction

The necessity of iron for the growth and virulence of the anthrax pathogen justifies the presence of a complex system for the assimilation of this trace element in Bacillus anthracis, which is not readily available in its free form.

One of the components of this system, functioning at the intracellular stage of anthrax infection, is represented by two high-affinity chelating agents—siderophores: petrobactin (PB) and bacillibactin (BB). The mutant strain ΔasbA with a deleted PB gene demonstrates a significantly weakened growth in macrophages and reduced virulence to mice, which gives reason to consider this siderophore as a minor (additional) virulence factor [1–4].

The second component of the iron uptake system uses hemoglobin heme as its source, acts in the extracellular stage of infection when the pathogen enters the bloodstream, and is carried out by heme-binding proteins. B. anthracis hemochromes include the Isd protein cluster: IsdX1 and IsdX2, IsdC, IsdA1, IsdA2, IsdE, IsdF, SrtB, IsdG, which degrade hemoglobin and transport heme. Furthermore, two intracellular proteins, HmoA and HmoB, which degrade heme, have been characterized, and it has been shown that the combined loss of HmoA and IsdG renders B. anthracis incapable of causing infection [5–9].

The functioning of the components of the iron uptake system in B. anthracis at two stages of anthrax infection is illustrated in Fig. 1.

 

Fig. 1. Model of the use of two alternative iron acquisition systems employed by B. anthracis during infection (adapted from [19]).

a — intracellular iron acquisition: I — spores enter the lungs to initiate the inhalation form of anthrax, where they are phagocytosed by resident phagocytes; II — during this initial intracellular phase, vegetative cells of B. anthracis synthesize and secrete two siderophores — PB and BB; III — these siderophores chelate phagocytic iron stores for use during infection. b — extracellular acquisition of heme iron: I — after B. anthracis emerges for replication in the extracellular environment, erythrocytes are lysed by hemolysins and cytolysins; II— hemoglobin is released and represents a rich iron store that can be targeted by B. anthracis; III — then heme is absorbed under the action of NEAT proteins, iron porphyrin is transported into the cell, and the released iron is used to stimulate bacterial replication.

 

The heme-binding protein Hal is considered to be an additional factor in B. anthracis responsible for iron uptake from heme. Its gene is thought to encode the N-terminal domain of the iron transporter (N-terminal near-iron transporter, NEAT), several internal regions rich in leucine repeats, and a C-terminal sortase-like domain. Deletion of its gene resulted in the microbe being unable to grow efficiently in a medium with heme or hemoglobin as an iron source [7].

Three processes must occur in order to access and utilize the host's hemoglobin as an iron source. First, red blood cells must be lysed by specific bacterial lipases, releasing hemoglobin into the plasma. Second, heme must be extracted from circulating hemoglobin and delivered to the bacterial cell envelope. Finally, heme must be transported through the envelope into the cell for degradation of its porphyrin and release of iron. The latter two processes are carried out by the cooperative action of the Isd system hemophores (IsdX1 and IsdX2, IsdC, IsdE, IsdF, IsdG), NEAT-containing Hal and Bslk proteins, and the intracellular heme-degrading monooxygenases HmoA and HmoB.

Evidence is provided that the B. anthracis S-layer protein (BslK), which is homologous to S-layer and NEAT, is localized on the cell surface and binds and transports heme to IsdC in a rapid, contact-dependent manner. These results show that the Isd system is not a self-sufficient channel for heme transport and imply that there is a functional cross-talk between differentially localized NEAT proteins that facilitates heme uptake during infection [10].

Hal and IsdX2, secreted by NEAT-containing hemophores, remove heme from host hemoglobin and transfer it to BslK. Further transport of heme across the bacterial envelope involves other NEAT-containing proteins, such as IsdX1 and IsdC, which are anchored by sortase B to the peptidoglycan of B. anthracis. Heme is imported into the bacterial cytoplasm by the ATP-binding protein IsdE and the membrane transporter IsdF. The heme oxygenases IsdG, HmoA, and HmoB cleave the tetrapyrrole moiety of heme, and the released iron is incorporated into iron-containing proteins [11].

Although heme is a valuable source of iron for B. anthracis during infection, heme and hemin (the free form of heme Fe(III) found in solution) can catalyze the formation of reactive oxygen species, leading to damage to proteins and lipids, causing the death of bacterial cells. The acquisition of iron from the host's heme can lead to the accumulation of toxic amounts of iron in B. anthracis. The HssRS sensor system, which regulates the expression of the heme-detoxifying transporter HrtAB, is designed to counteract the toxicity of heme. It includes the membrane-localized histidine kinase HssS, which senses heme in an unknown manner and undergoes histidine phosphorylation. HssR is a cytoplasmic response regulator that recognizes phosphorylated HssS and catalyzes the transfer of phosphorus to the conserved aspartic acid residue of HssR. When phosphorylated, HssR binds to a direct repeat in the hrtAB promoter, causing its expression and export from the cytoplasm of excess heme or toxic metabolites accumulated in the cell under its action [12].

FapR, a transcriptional regulator of fatty acid biosynthesis, has been identified as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and HssS localization. Disruption of fapR leads to increased membrane rigidity, hindering the penetration of HssRS inducers, which causes its inactivation. Deletion of fapR disrupts the function of HssS sensitivity to heme and reduces endogenous heme biosynthesis. FapR appears to be a potential target for the development of antibacterial agents [13].

The toxic effects of iron in B. anthracis are also neutralized by the immunogenic proteins Dlp-1 and Dlp-2, whose genes are homologous to the genes of the DNA-binding proteins Dps1 and Dps2. Both are capable of binding and isolating free iron, which allows bacteria to grow in iron-overloaded conditions by reducing the amount of free iron in the culture medium. Overall, these data indicate that Dlp-1 and Dlp-2 act as ferritins in B. anthracis [14]. It is assumed that these paired proteins in B. anthracis perform different functions: Dlp-1 (Dps1) sequesters iron, while Dlp-2 (Dps2) causes the destruction of hydrogen peroxide [15].

We have previously identified significant differences in the structure of genes and proteins associated with spore germination, sporulation, and virulence in strains of B. anthracis from major genetic lineages, which may influence their adaptation and prevalence [16–18]; however, the characteristics of the iron uptake system were not considered in these studies.

To date, all data on the influence of various components of the iron uptake system on the viability and virulence of B. anthracis have been obtained in experiments with variants in which the function is disabled due to mutations in the corresponding genes. Meanwhile, differences in their virulence and prevalence may be associated, among other things, with the genetic characteristics of siderophores and hemophores. To date, nothing is known about the genetic variability of the components of the iron uptake system of natural strains of B. anthracis, which makes this study relevant.

The aim of this study is to characterize the genes and proteins of the iron uptake system in natural strains of B. anthracis of different genetic lineages.

Materials and methods

This study examined 937 complete genomes of B. anthracis strains from the NCBI GenBank database, belonging to 14 canSNP groups of the main genetic lineages A (839 strains), B (93 strains), C (5 strains), and the genomes of 4 strains of B. cereus biovar anthracis, taken for comparison.

We also analyzed 10 complete genomes of B. anthracis strains from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor, selected on the basis of their belonging to 5 canSNP groups found in Russia, Georgia, and Azerbaijan (Table 1).

 

Table 1. Strains of B. anthracis from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor

No.	Strain	Year of isolation	Place of isolation	Source of isolation	canSNP-group
1	И-45 (I-45)	1966	Russia	Soil	A.Br.001/002
2	И-271 (I-271_OBL)	2016	Russia	Deer carcass	A.Br.001/002
3	351-520	1976	Azerbaijan	Soil	A.Br.Aust94
4	737-10	1984	Georgia	Human	A.Br.Aust94
5	И-319 (I-319)	1983	Russia	Human	A.Br.Ames
6	И-360 (I-360)	2006	Russia	Human	A.Br.Ames
7	1390-13	2022	Russia	Ulcer of patient	A.Br.008/011
8	81-1	1969	Russia	Ulcer of patient	A.Br.008/011
9	228	1946	Georgia	Sheep carcass	B.Br.001/002
10	1284	2010	Russia	Dumplings	B.Br.001/002

 

Genomic sequences of B. anthracis strains from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor are presented in the electronic database "Nucleotide sequences of complete genomes of B. anthracis strains isolated in Russia and neighboring countries" (Certificate of State Registration No 2022620144 dated 01/18/2022), in the electronic database of the Stavropol Anti-Plague Institute of Rospotrebnadzor "Whole-genome sequences of B strains. anthracis" (reg. No B.ab-R-1 – B.ab-R-302) and can be provided upon request (stavnipchi@mail.ru). The genomic sequence of the B. anthracis 81/1 (81-1) strain was also deposited in GenBank with access number CA_003860145.1

Genome analysis was performed in silico using the genome of B. anthracis Ames Ancestor (GenBank: NC_007530.2; NC_007322.2; NC_007323.3) as a reference strain (RS). Gene and protein sequence alignment and nucleotide translation were performed using MEGA X software. Proteins were verified using the NCBI Protein Database. Polymorphisms were identified using BLASTn, BLASTp, and MEGA X software. The presence of single nucleotide polymorphisms (SNPs), amino acid substitutions (AAS), insertions/deletions (INDELs), and the number of variants for each protein were evaluated.

Results

Data on the variability of siderophores and hemophores are presented in Table 2. The localization and nature of SNPs in B. anthracis are summarized in Table 3.

 

Table 2. Polymorphisms in siderophores and hemophores of B. anthracis and B. cereus biovar anthracis

Protein/Gene	Main lineages
	A				B				C				B. cereus biovar anthracis
	Polymorphisms
	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants
Siderophores
DhbA(EntA)(2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase)	11	0	8	5	1	0	1	1	2	0	2	1	4	0	2	1
DhbB(isochorismatase)	2	0	2	2	1	0	1	2	0	0	0	1	17	0	8	1
DhbC (EntC) (isochorismate synthase)	31	1	15	8	0	0	0	1	0	0	0	1	20	0	5	1
DhbE (2,3-dihydroxybenzoate-AMP ligase, 2,3-dihydroxybenzoyl)adenylate synthase )	5	0	5	6	3	0	2	2	0	0	0	1	12	0	3	1
DhbF(nonribosomal peptide synthetase DhbF)	13	2	9	12	2	1	1	0	0	0	0	1	207	0	81	1
MbtH (MbtH family protein, iron compound ABC transporter)	2	0	1	3	0	0	0	1	0	0	0	1	1	0	0	1
MDR family MFS transporter (drug resistance transporter)	15	0	12	6	0	0	0	1	0	0	0	1	6	0	2	1
Sfp(4'-phosphopantetheinyl transferase Sfp)	14	0	7	5	0	0	0	1	0	0	0	1	5	0	1	1
UbiC(UbiC-like conserved hypothetical protein)	2	0	2	3	1	0	1	2	0	0	0	1	0	0	0	1
Yuil alpha/beta hydrolase	5	0	4	4	1	0	1	1	1	0	1	1	9	0	3	1
Iron compound ABC transporter FeuD	7	0	3	4	0	0	0	1	1	0	1	1	6	0	3	1
FecCD family ABC transporter permease	1	0	1	2	1	0	1	2	0	0	0	1	6	0	2	1
FeuB iron compound ABC transporter, permease protein	14	0	7	5	1	0	1	1	1	0	1	1	5	0	4	1
Petrobactin biosynthesis protein AsbA	11	0	11	6	0	0	0	1	0	0	0	1	7	0	3	1
Petrobactin biosynthesis protein AsbB	5	0	5	4	0	0	0	1	1	0	1	2	17	0	9	1
Petrobactin biosynthesis protein AsbC	2	0	2	3	0	0	0	1	3	0	2	2	16	0	8	1
Petrobactin biosynthesis protein AsbD	0	0	0	1	0	0	0	1	0	0	0	1	0	0	0	1
Petrobactin biosynthesis protein AsbE	25	0	11	2	0	0	0	1	2	0	1	2	4	0	1	1
Petrobactin biosynthesis protein AsbF	1	0	1	2	0	0	0	1	0	0	0	1	0	0	0	1
FhuB-like iron compound ABC transporter, permease protein	3	0	3	4	0	0	0	1	2	1	2	2	0	0	0	0
FpuA iron compound ABC transporter	0	0	0	1	0	0	0	1	0	0	0	1	0	0	0	0
FatB iron compound ABC transporter, iron compound-binding	11	1	7	3	0	0	0	1	0	0	0	1	50	1	11	1
FatC iron compound ABC transporter, permease protein	4	0	3	3	1	0	1	2	1	0	1	1	8	0	3	1
FatD iron compound ABC transporter, permease protein	2	1	2	3	1	1	1	2	2	1	2	1	40	1	11	1

 

End of the Table 2.

Protein/Gene	Main lineages
	A				B				C				B. cereus biovar anthracis
	Polymorphisms
	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants	SNP	INDEL	Amino acid substitutions	Protein variants
FhuC-like iron compound ABC transporter, ATP-binding protein	6	0	3	4	0	0	0	1	0	0	0	1	21	0	3	1
Total	192	5	124		13	2	11		16	2	14		461	2	163
Per 1 genome	0,226	0,006	0,146		0,137	0,021	0,116		3,2	0,4	2,8		115,25	0,500	40,75
Hemophores
IsdC Heme uptake protein	2	0	2	3	0	0	0	1	0	0	0	1	70	1	17	1
IsdE Heme_ABC transporter substrate binding protein	11	0	6	5	0	0	0	1	0	0	0	1	22	1	5	1
IsdG Heme_oxygenase	1	0	1	2	0	0	0	1	0	0	0	1	0	0	0	1
IsdE2– FecCD family ABC transporter permease	23	0	5	4	0	0	0	1	0	0	0	1	38	0	8	1
IsdF ABC transporter ATP binding protein	1	0	1	2	0	0	0	1	1	0	1	2	48	0	11	1
IsdX1– NEAT domain containing protein	0	0	0	1	0	0	0	1	0	0	0	1	0	0	0	0
IsdX2 – NEAT domain containing protein	7	0	3	4	1	2	1	1	0	0	0	0	0	0	0	0
SrtB MULTISPECIES_class B sortase	1	0	1	2	0	0	0	1	1	0	0	1	7	0	2	1
HmoA heme-degrading monooxygenase	1	0	1	2	0	0	0	1	0	0	0	1	5	0	0	1
HmoB heme-degrading monooxygenase	1	0	1	2	0	0	0	1	0	0	0	1	8	2	1	1
Dlp-1 (Dps-1) DNA protection during starvation protein	0	0	0	1	0	0	0	1	0	0	0	1	1	0	0	1
Dlp-2 (Dps-2) DNA protection during starvation protein	2	0	2	3	0	0	0	1	0	0	0	1	1		1	1
Hal leucine-rich repeat domain-containing protein	4	0	3	4	7	2	0	2	1	1	0	1	0	0	0	0
HtrA_MULTISPECIES_ABC transporter ATP-binding protein	1	1	1	2	0	0	0	1	0	0	0	1	16	0	2	1
HtrB_ABC transporter permease_	1	0	1	2	1	0	1	1	0	0	0	1	25	0	6	2
HssR_MULTISPECIES_sensor histidine kinase	0	0	0	1	0	0	0	1	0	0	0	1	62	0	8	1
HssS_MULTISPECIES_response regulator transcription factor	0	0	0	1	0	0	0	1	0	0	0	1	30	0	1	1
BslK S-layer homology domain-containing protein	1	1	1	3	1	0	1	1	1	0	1	1	30	1	9	1
Total	53	2	29		10	4	3		3	2	2		363	5	71
Per 1 genome	0,060	0,02	0,034		0,0116	0,042	0,031		0,6	0,4	0,4		121	1,66	23,6

 

Table 3. The nature of single nucleotide polymorphisms in siderophores and hemophores of B. anthracis

Protein/Gene	Main lineages
	A			B			C
	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution
Siderophores
DhbA(EntA)(2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase)	35T→C 291A→G 762C→T	34T→G 58A→T 183A→T 212A→G 226A→C 232A→C 244C→T 685A→G	L12A I20L L61F D71G I76L N78H P82S T229A	None	773T→C	V258A	None	685A→G 754G→A	T229A G252S
DhbB(isochorismatase)	None	53C→T 880G→A	P18L V294L	None	None	None	None	None	None
DhbC (EntC) (isochorismate synthase)	78T→C 219G→T 276G→C 348G→A 354G→A 462T→C 486T→G 568A→G 597T→C 660C→T 750T→C 756G→A 952C→G 955A→C 999T→G 1106C→G	62A→C 86C→A 301C→A 332T→C 378A→T 392C→T 464G→A 516C→A 571T→C 649C→A 781T→C 947C→T 954A→T 1103A→T 1107A→T	E21A P29Q P101T L111S K126N G131V G155E H172Q T190A P217T Y261H A316V Q318D E368V S369C	None	None	None	None	None	None
DhbE (2,3 dihydroxybenzoate AMP ligase, 2,3 dihydroxybenzoyl)adenylate synthase)	None	307G→A 859C→A 1451C→T 1483C→T 1558G→C	V103I L287I A484V R495C V520L	1392T→C	59G→A 554C→T	G20E A185V	None	None	None
DhbF(nonribosomal peptide synthetase DhbF)	2673A→N 4578A→G 5105G→A 5465G→N	1180C→T 1810G→A 2105T→C 2111T→C 5077G→T 5522C→T 5615T→C 6520C→T 6751A→T	H394Y E604K M702T A704V V1693L S1841F V1872A L2174F I2251F	4794A→G	6252G→A	M2084I	None	None	None

 

Continuation of the Table 3.

Protein/Gene	Main lineages
	A			B			C
	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution
MbtH (MbtH family protein, iron compound ABC transporter)	198C→G	206T→A	I69K	None	None	None	None	None	None
MDR family MFS transporter (drug resistance transporter)	279G→T 288C→T 516A→G	25G→T 184C→T 282A→G 290A→G 305A→T 316A→G 329C→T 458T→A 497C→T 641C→T 1204G→A 1366G→T	V9L L62F I94M D97G N102I I106V A110V I153N A166V A214V G402S D456Y	None	None	None	None	None	None
Sfp(4'phosphopantetheinyl transferase Sfp)	120A→G 382C→A 392C→A 399G→A 444A→G 486T→G 520C→T	91T→A 161C→T 316T→C 378T→G 393G→A 601G→T 679A→G	W31R A54V S106P Q128K P131Q V201F I227V	None	None	None	None	None	None
UbiC(UbiC-like conserved hypothetical protein)	None	380A→G 419A→G	E127G D140G	None	511C→G	H171D	None	None	None
Yuil alpha→beta hydrolase	249T→C	281G→A 319T→A 646A→G 761A→T	G94D S107T S216G H254L	None	646A→G	S216G	None	646A→G	S216G
Iron compound ABC transporter FeuD	129A→G 186A→G 240C→G 246C→T	220C→G 454G→A 565C→T	Q74E V152M H189Y	None	None	None	None	454G→A	V152M
FecCD family ABC transporter permease	None	118G→A	V40I	None	854G→A	Q285R	None	None	None
FeuB iron compound ABC transporter, permease protein	18G→A 30C→T 45A→G 714G→A 732T→A 750G→A 867T→C	106G→A 230C→T 281T→G 310G→A 464T→G 490A→G 626T→C	A36T S77L I94S V104I V155G D164G V209A	None	626T→C	V209A	None	626T→C	V209A

 

Continuation of the Table 3.

Protein/Gene	Main lineages
	A			B			C
	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution
Petrobactin biosynthesis protein AsbA	None	407T→A 470T→C 511C→T 557C→A 559C→A 562G→A 578G→C 625T→C 656T→C 728A→T 1760A→G	F136Y A157V P171S S186Y P187T E188K C193S S209P L219S Y243F Y587C	None	None	None	None	None	None
Petrobactin biosynthesis protein AsbB	None	97T→A 106T→A 783T→A 1187G→ 1608G→A	S33T Y36N D261E G396E M536I	None	None	None	None	928C→T	R310C
Petrobactin biosynthesis protein AsbC	None	626C→T 1063A→T	P209L I355F	None	None	None	231T→C	398T→C 1056G→A	V133A M352I
Petrobactin biosynthesis protein AsbD	None	None	None	None	None	None	None	None	None
Petrobactin biosynthesis protein AsbE	303C→A 495A→G 843A→G 906A→G 912C→T 916A→G 917C→A 933G→A 937T→C 942A→T 950A→C 958T→A 973T→G 977T→C	370T→A 382G→A 918T→G 929G→A 951A→T 954G→A 959C→T 964G→C 976G→A 978A→T 979T→C	L124I E128K T306E R310K K317A M318I S320I E322Q L325V V326T F327L	None	None	None	303C→A	410T→C	M137T
Petrobactin biosynthesis protein AsbF	None	680T→C	V227A	None	None	None	None	None	None
FhuB-like iron compound ABC transporter, permease protein	None	308A→G 617T→C 1234G→T	I103C F206S A412S	None	None	None	None	1379C→A 1798G→A	P460H G600S
FpuA iron compound ABC transporter	None	None	None	None	None	None	None	None	None

 

Continuation of the Table 3.

Protein/Gene	Main lineages
	A			B			C
	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution
FatB iron compound ABC transporter, iron compound binding	151C→A 166G→A 408C→A 415T→C	153T→A 160G→A 168T→C 173T→C 500T→G 512C→A 389C→A	H51K V54I V56I V58A L167R T171K A130D	None	None	None	None	None	None
FatC iron compound ABC transporter, permease protein	666T→A	659T→C 671T→A 913C→T	V220A 1224N P305S	None	250A→G	I84V	None	829A→G	I277V
FatD iron compound ABC transporter, permease protein	None	490G→A 623C→T	V164I T208I	None	448T→G	S150A	None	316A→G 931C→G	S106G P311A
FhuC-like iron compound ABC transporter, ATP binding protein	114T→A 130C→A 134T→C	121T→A 132T→G 742G→A	S41T L44M A248T	None	None	None	None	None	None
Hemophores
IsdC Heme uptake protein	None	329C→T 365C→A	T110I A122E	None	None	None	None	None	None
IsdE Heme_ABC transporter substrate binding protein	489A→G 579G→A 766T→A 777T→A 792T→A	19G→A 34G→A 164T→C 212A→G 769G→C 771T→A	A7T A12T L55S K71R F256I A257P	None	None	None	None	None	None
IsdG Heme_oxygenase	None	19A→T	T7S	None	None	None	None	None	None
IsdE2 – FecCD family ABC transporter permease	70A→G 144A→G 147G→C 195A→C 243T→C 249C→T 261A→G 264G→A 267T→C 381C→T 627T→C 634T→A 701T→C	198G→T 215G→A 232G→T 448A→C 636A→G	A66G G72D V78F N150H S212T	None	None	None	None	None	None

 

End of the Table 3.

Protein/Gene	Main lineages
	A			B			C
	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution	SNP synonymous	SNP non-synonymous	Amino acid substitution
	714C→T 768T→C 774T→G 786A→G 924T→G
IsdF ABC transporter ATP binding protein	None	257C→A	A86E	None	None	None	448A→G	M150V
IsdX1 – NEAT domain containing protein	None	None	None	None	None	None	None	None	None
IsdX2 – NEAT domain containing protein	284C→T 586A→G 743A→G 2621A→G	93C→T 800A→G 976G→A	N196D K267R G326S	None	None	None	None	None	None
SrtB MULTISPECIES_class B sortase	None	32T→A	I11N	None	None	None	219G→T	None	None
HmoA heme degrading monooxygenase	None	19A→G	T19A	None	None	None	None	None	None
HmoB heme degrading monooxygenase	None	155G→C	G52A	None	None	None	None	None	None
Dlp1 (Dps1) DNA protection during starvation protein	None	None	None	None	None	None	None	None	None
Dlp2 (Dps2) DNA protection during starvation protein	None	235A→G 365C→A	T79A A122E	None	None	None	None	None	None
Hal leucine rich repeat domain containing protein	2654C→T	742A→G 767C→T 2550A→C	K248E S256F E850D	744 A→G 2670A→C 2671T→C 2685A→G 2712C→T	2673A→G 2695A→G	S891P A899T	2817T→A	None	None
HtrA_MULTISPECIES_ABC transporter ATP binding protein	None	370C→T	К124С	None	None	None	None	None	None
HtrB_ABC transporter permease_	None	575C→T	P192L	None	259G→A	A87T	None	None	None
HssR_MULTISPECIES_sensor histidine kinase	None	None	None	None	None	None	None	None	None
HssS_MULTISPECIES_response regulator transcription factor	None	None	None	None	None	None	None	None	None
BslK S layer homology domain containing protein	None	218T→C	L73P	None	218T→C	L73P	None	None	None

 

Genes encoding enzymes involved in BB biosynthesis, entA–dhbBCF cluster [2, 4]

• DhbA(EntA)(2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase). Strains of anthracis from the main genetic lineage A had 5 variants of DhbA. Strains from lineage B had 1 variant with 1 SNP and 1 AAS. Strains from lineage C had another variant with 2 non-synonymous SNPs. The third variant, with 2 SNPs and 2 AAS, was found in the B. cereus biovar anthracis CI strain.
• DhbB isochorismatase. Two variants were found in anthracis strains of lineage A: the RS variant with 2 SNPs and 2 AAS. Two variants were also found in strains of lineage B: variant RS; with 1 SNP and 1 AAS. Strains of lineage C had variant RS. Strains of B. cereus biovar anthracis had a variant with 17 SNPs and 8 AAS.
• DhbC isochorismatase. In strains of lineage A — 8 variants with 31 SNPs, 15 AAS, and 1 indel. In strains of lineages B and C — variant RS. In strain cereus biovar anthracis — variant with 20 SNPs and 5 AAS.
• Dehydrobenzoate ATP ligase DhbE. In strains of anthracis lineage A, there are 6 variants with 5 SNPs and 5 AAS. In strains of lineage C, there is the RS variant. Strains of lineage B have 2 variants: with 3 SNPs and 2 AAS. In the variant of B. cereus biovar anthracis strains, there are 12 SNPs and 3 AAS.
• Non-ribosomal peptidyl-tRNA synthetase DhbF. In strains of lineage A, there are 12 variants with 13 SNPs, 9 AAS, and 2 indels. In strains of lineage C, there is the RS variant. In strains of cereus biovar anthracis, there is the largest set of polymorphisms, with 207 SNPs and 81 AAS. In strains of lineage B, dhbF, with 2 SNPs and 1 AAS, is a pseudogene due to the deletion of G2415 and a reading frame shift, resulting in the absence of a functional protein (Fig. 2).

 

Fig. 2. Features of the dhbF gene of strain SVA11 of the main lineage B.

a — gene alignment; b — alignment of the translated gene sequence; c — fragment of the gene description from the complete genome sequence in GenBank NCBI Reference Sequence NZ_CP006742.1

 

• ABC iron compound transporter. Strains of lineage A have 3 variants — the RS variant and 2 more with 2 SNPs, 1 AAS. Strains of lineages B and C have the RS variant. Strains of cereus biovar anthracis have a variant with 1 synonymous SNP.
• MFS efflux transporter. Strains of anthracis lineage A have 6 variants with 15 SNPs and 12 AAS. Strains of lineages B and C have the RS variant. Strains of B. cereus biovar anthracis have a variant with 6 SNPs and 2 AAS.
• 4'-phosphopantetheinyl transferase Sfp. In strains of lineage A, there are 5 variants with 14 SNPs and 7 AAS. In strains of anthracis lineages B and C, there is the RS variant. In strains of B. cereus biovar anthracis, there is a variant with 5 SNPs and 1 AAS.
• Homolog of UbiC, encoding chorismate pyruvate lyase. In strains of anthracis lineage A, there are 3 variants with 2 SNPs and 2 AAS. In strains of lineage B, there are 2 variants: the RS variant and a variant with 1 SNP and 1 AAS. In strains of B. anthracis lineage C and strains of B. cereus biovar anthracis, there is the RS variant.

Genes of enzymes involved in BB absorption, FEUABCD/YUII cluster

• Alpha-beta hydrolase Yuil. In anthracis lineage A, there are 4 variants with 5 SNPs and 4 AAS. In B. anthracis strains of lineages B and C, there is 1 variant with 1 SNP and 1 AAS. In B. cereus biovar anthracis strains, there is 1 variant with 9 SNPs and 3 AAS.
• ABC iron compound transporter, ATP-binding protein FeuD. B. anthracis strains of lineage A have 4 variants with 7 SNPs and 3 AAS. anthracis strains of line B have 1 variant of RS. Strains of lineage C have 1 variant with 1 SNP and 1 AAS. In B. cereus biovar anthracis strains, there is 1 variant with 6 SNPs and 3 AAS.
• ABC transporter permease of the FecCD family. In anthracis strains of lineages A and B, there are 2 variants each: 1 variant of RS; with 1 SNP and 1 AAS. In strains of B. anthracis lineage C — RS variant. In strains of B. cereus biovar anthracis — 1 variant with 6 SNPs and 2 AAS.
• ABC transporter permease of the FeuB family. In strains of anthracis lineage A — 5 variants with 14 SNPs and 7 AAS. In strains of lineages B and C, there is 1 variant with a non-synonymous SNP and AAS. In strains of B. cereus biovar anthracis, there is 1 variant with 5 SNPs and 4 AAS.
• The iron-binding ABC transporter FeuA in B. anthracis is degenerate, as the pseudogene contains a reading frame shift.
• The only enzyme associated with BB export, YmfD, is also degenerate in anthracis, as the chromosome region is represented by a pseudogene and contains one or more premature stop codons and a reading frame shift.

Genes of enzymes involved in PB biosynthesis, Asb cluster

• PB AsbA biosynthesis protein. B. anthracis strains of lineage A have 6 variants: 1 variant of RS and 5 variants with 11 SNPs and 11 AAS. anthracis strains of lineages B and C have 1 variant of RS. B. cereus biovar anthracis strains have 1 variant with 7 SNPs and 3 AAS.
• PB biosynthesis protein AsbB: 4 variants for anthracis lineage A with 5 SNPs and 5 AAS. In B. anthracis lineage B, there is 1 variant of RS. In B. anthracis lineage C, there are 2 variants: 1 variant of RS, with 1 SNP and 1 AAS. In B. cereus biovar anthracis strains — 1 variant with 17 SNPs and 9 AAS.
• PB biosynthesis protein AsbC. In anthracis strains of lineage A — 3 variants: one RS variant and 2 variants with 2 SNPs and 2 AAS. In strains of lineage B, there is 1 variant of RS. In strains of lineage C, there are 2 variants with 3 SNPs and 2 AAS. In strains of B. cereus biovar anthracis, there is 1 variant with 16 SNPs and 8 AAS.
• PB biosynthesis protein AsbD. In strains of anthracis of all 3 lineages and strains of B. cereus biovar anthracis — 1 variant of RS.
• PB biosynthesis protein AsbE. In anthracis strains of lineage A — 2 variants: variant RS; with 25 SNPs and 11 AAS. In strains of lineage B, there is one variant of RS. In strains of B. anthracis lineage C, there are two variants: one variant of RS; with 2 SNPs and 1 AAS. In strains of B. cereus biovar anthracis, there is one variant with 4 SNPs and 1 AAS.
• PB biosynthesis protein AsbF. In anthracis strains of lineage A, there are 2 variants: RS variant; with 1 SNP and 1 AAS. In strains of B. anthracis lineages B, C, and B. cereus biovar anthracis, there is 1 RS variant.

Genes of enzymes involved in PB biosynthesis, FPUA/FHUB cluster

• FhuB-like ABC transporter of iron compounds, permease protein. In anthracis strains of lineage A, there are four variants: the RS variant and three variants with three SNPs and three AAS. In strains of lineage B, there is one variant of RS. In strains of lineage C, there are two variants with two SNPs and two AAS and a triplet deletion in the region with three tandem repeats. Not identified in strains of B. cereus biovar anthracis.
• ABC iron compound transporter, FpuA permease. In strains of anthracis of all 3 lineages — 1 variant. Not identified in strains of B. cereus biovar anthracis.

Genes of enzymes involved in PB biosynthesis, FATBCD/FHUC cluster

• ABC iron compound transporter, FatB permease. In anthracis strains of lineage A, there are three variants: RS variant; two variants with one insertion, 11 SNPs, and seven AAS. In B. anthracis strains of lineages B and C. In B. cereus biovar anthracis strains, there is 1 variant with 50 SNPs, 11 AAS, and 1 insertion.
• ABC iron compound transporter, FatC permease. In anthracis strains of lineage A, there are 3 variants: the RS variant; 2 variants with 4 SNPs and 3 AAS. In strains of B. anthracis lineage B, there are 2 variants: RS variant; with 1 SNP and 1 AAS. In strains of lineage C, there is 1 variant with 1 SNP and 1 AAS. In strains of B. cereus biovar anthracis, there is 1 variant with 8 SNPs and 3 AAS.
• ABC iron compound transporter, FatD permease. In anthracis strains of lineage A, there are 3 variants: the RS variant and 2 variants with 1 insertion, 2 SNPs, and 2 AAS. In strains of lineage B, there is 1 variant with 1 insertion, 1 SNP, and 1 AAS. Lineage C strains have 1 variant with 1 insertion, 2 SNPs, and 2 AAS. B. cereus biovar anthracis has 1 variant with 1 insertion, 40 SNPs, and 11 AAS. Insertion 1–12 is present in all variants.
• ABC iron compound transporter binding ATP FhuC-like protein. In strains of anthracis lineage A, there are 4 variants: the RS variant, 3 variants with 6 SNPs, and 3 AAS. In lineages B and C, there is 1 RS variant. In B. cereus biovar anthracis, there is 1 variant with 21 SNPs and 3 AAS.

Enzymes involved in the biosynthesis, assembly, and absorption of hemochromes

• Heme-binding protein IsdC. In anthracis strains of lineage A, Heme-binding protein IsdC there are three variants: the RS variant and two variants with two SNPs and two AAS. In lineages B and C, there is one RS variant. In B. cereus biovar anthracis, there is one variant with 70 SNPs, 17 AAS, and one insertion.
• ABC heme transporter substrate-binding protein IsdE. In anthracis strains of lineage A, there are five variants: the RS variant and four variants for four strains with 11 SNPs and five AAS. In lineages B and C, there is one RS variant. In B. cereus biovar anthracis there is 1 variant with 22 SNPs, 5 AAS, and 1 insertion.
• Heme oxygenase IsdG. In anthracis strains of lineage A — 2 variants: variant RS; with 1 SNP and 1 AAS. In strains of B. anthracis lineages B, C, and in strains of B. cereus biovar anthracis — 1 variant of RS.
• ABC transporter permease of the FecCD family IsdE2. In strains of anthracis lineage A — 4 variants: variant RS and 3 variants with 22 SNPs and 5 AAS. In lineages B and C, there is 1 variant of RS. In strains of B. cereus biovar anthracis, there is 1 variant with 38 SNPs and 8 AAS.
• ABC transporter binding ATP protein IsdF. In strains of anthracis lineage A, there are 2 variants: variant RS; with 1 SNP and 1 AAS. In strains of B. anthracis lineage B, there is 1 variant of RS. In strains of B. anthracis lineage C, there are 2 variants: variant RS; with 1 SNP and 1 AAS. In strains of B. cereus biovar anthracis, there are 48 SNPs and 11 AAS.
• IsdX1 protein containing the NEAT domain. One RS in all three anthracis strains. Not identified in B. cereus biovar anthracis strains.
• IsdX2 protein containing the NEAT domain. Within IsdX2, a VNTR with a repeat unit of 15 bp GAAGTAGAAAAACCA and a number of units from 1 to 6 has been identified. In anthracis strains of lineage A, there are 4 variants: the RS variant and 3 variants with 3 SNPs and 3 AAS. In B. anthracis strains of lineage C, there is one PS variant. In B. anthracis strains of lineage B, there is one variant with two insertions and one SNP. In B. cereus biovar anthracis strains, it has not been identified.
• Class B sortase SrtB. In strains of anthracis lineage A, there are 2 variants differing by 1 SNP and 1 AAS. In strains of lineage B, there is 1 variant of RS. In strains of lineage C, there is 1 variant with 1 SNP. In strains of B. cereus biovar anthracis, there is 1 variant with 7 SNPs and 2 AAS.

Intracellular proteins that degrade heme

• Degrading heme monooxygenase HmoA. anthracis strain A has two variants, one of which differs from RS, 1 SNP and 1 AAS. B. anthracis strains B, C, and B. cereus biovar anthracis strains have one variant of RS, while the B. cereus biovar anthracis gene has five synonymous SNPs.
• Degradative heme monooxygenase HmoB. In anthracis strain A, there are two variants: RS; with 1 SNP and 1 AAS. In B. anthracis strains B and C, there is one RS variant. In B. cereus biovar anthracis strains, there is a variant with 2 deletions, 8 SNPs, and 1 AAS.

Proteins associated with iron acquisition, whose genes are homologous to DNA-binding protein genes

• Dlp-1 (Dps-1) protein, which protects DNA during starvation. All strains of anthracis have 1 variant; B. cereus biovar anthracis has 1 synonymous SNP.
• Dlp-2 (Dps-2) protein, which protects DNA during starvation. In anthracis strains of lineage A, there are 3 variants: RS variant; 2 variants with 2 SNPs and 2 AAS. In B. anthracis strains of lineages B and C, there is 1 RS variant. In B. cereus biovar anthracis strains, there is 1 variant with 1 SNP and 1 AAS.
• Hal repeat domain containing protein. In anthracis strains of lineage A, there are 4 protein variants: the RS variant; 3 variants with 4 SNPs and 3 AAS. In B. anthracis strains of lineage B, there are 2 variants with 7 SNPs and 2 AAS. In B. anthracis strains of lineage C, there is 1 variant with 1 insertion and 1 SNP. In B. cereus biovar anthracis strains, no variants have been identified.

Heme resistance enzymes and their regulation

• ABC transporter ATP binding protein HtrA. Strains of lineage A have two variants: RS variant; with 1 SNP, 1 AAS, and 1 insertion. Lineages B and C have the RS variant. cereus biovar anthracis has a variant with 16 SNPs and 2 AAS.
• ABC transporter permease HtrB. Strains of anthracis lineage A have two variants: variant RS; 1 SNP and 1 AAS. Strains of lineage B have one variant with 1 SNP and 1 AAS. In strains of lineage C, there is 1 RS variant. In strains of B. cereus biovar anthracis, there are 2 variants with 25 SNPs and 6 AAS.
• Sensory histidine kinase HssR. Identical in anthracis strains of all lineages. In B. cereus biovar anthracis, there is 1 variant with 62 SNPs and 8 AAS.
• Transcription regulatory response factor HssS. Identical in anthracis strains of all lineages. In B. cereus biovar anthracis, there is 1 variant with 30 SNPs and 1 AAS.
• BslK S layer homology domain containing protein. In anthracis strains of lineage A, there are 3 variants: 1 RS variant; 1 variant identical to strains of lineages B and C, with 1 SNP and 1 AAS; 1 variant in strain A 34F2 Sterne with 1 insertion and start codon TTG. In B. cereus biovar anthracis, there is a variant with a deletion, 30 SNPs, and 9 AAS.

Discussion

The iron acquisition system of B. anthracis involves 43 enzymes, whose genes are represented by a total of 51,157 nucleotides, i.e., about 1% of the entire genome. The biosynthesis, assembly, and absorption of siderophores are carried out by 25 enzymes, whose genes total 32,109 nucleotides, approximately 63% of all genes involved in the system, while the functioning of hemochromes, degrading hemoglobin, transporting heme, determining resistance to the toxic effects of heme, as well as ferritins, is supported by 18 enzymes, whose genes are represented by a total of 19,048 nucleotides, or 37% of all genes.

The abundance and diversity of siderophore and heme transporter genes indicates that iron acquisition is an important process during the reproduction of B. anthracis in the host organism [19].

The genetic variability of the components of the iron uptake system is expressed to varying degrees in strains of B. anthracis from three main genetic lineages.

The frequency of occurrence of all types of polymorphisms in siderophore proteins was comparable for B. anthracis strains of lineages A and B, while the DhbF non-ribosomal peptide synthetase protein in strains of lineage B was inactive. The frequency of polymorphisms in proteins of strains of lineage C was an order of magnitude higher than in lineages A and B, but significantly lower than in B. cereus biovar anthracis.

BB is synthesized in a manner typical for most macrolactone siderophores using nonribosomal peptide synthetases [20]. The non-ribosomal peptide synthetase DhbF, consisting of 2385 amino acids, in B. cereus biovar anthracis strains was radically different from the synthetase of B. anthracis, having the largest set of polymorphisms of 207 SNPs and 81 AAS. This protein was also the most variable in B. anthracis strains of lineage A, with 12 variants. The remaining proteins involved in BB biosynthesis and uptake in strains of lineage A had 2 to 8 variants. Lineage B strains had 1–2 variants of all proteins in this class, with the exception of DhbF, which was inactive due to a pseudogene. Lineage C strains had 1 variant of these proteins, with 3 non-synonymous SNPs. BB biosynthesis proteins had the most polymorphisms in B. cereus biovar anthracis strains, significantly fewer in B. anthracis lineage A strains, and the fewest in B. anthracis strains of lineages B and C. MbtH, a transport protein, was the most conservative of these, with one variant in strains of B. anthracis lineages B and C and B. cereus biovar anthracis, and two in strains of B. anthracis lineage A.

Of the 12 PB biosynthesis proteins in B. anthracis strains of lineage A, only AsbD and FpuA had 1 variant, and they were also invariable in B. anthracis strains of lineages B and C and in B. cereus biovar anthracis. In B. anthracis strains of lineage B, only FatC and FatD were represented by 2 variants.

The strains of B. anthracis lineage C had two variants each of the proteins AsbA, AsbB, AsbE, and FhuB-like. B. cereus biovar anthracis had one variant each with 163 SNPs, 49 AAS, and two indels; the proteins FhuB-like and FpuA could not be identified.

A comparison of the relative number of polymorphisms in siderophore genes per genome showed that B. cereus biovar anthracis strains had the highest number. This is explained by the special position of this subspecies, which has a chromosome of classical B. cereus representatives that differs significantly from the chromosome of B. anthracis.

Of the 18 heme-related proteins, as well as those associated with heme degradation, iron uptake, heme resistance, and their regulation, 4 proteins in B. anthracis strains of lineage A had no variants, while the rest had 2–5 variants. In B. anthracis strains of lineages B and C, 1 protein had 2 variants. The frequencies of polymorphisms were practically the same in B. anthracis strains of lineages A and B, while in strains of lineage C they were an order of magnitude higher. In B. cereus biovar anthracis, 3 proteins were not identified, while the others had 1 variant each, but the frequencies of polymorphisms were incomparably higher than in B. anthracis.

There were two variants in class B sortase in strains of lineages A and B and in intracellular proteins degrading heme monooxygenases HmoA and HmoB.

The most conservative genes and proteins in B. anthracis and B. cereus biovar anthracis are Dlp-1 and Dlp-2, which protect against the damaging effects of excess heme iron and toxic metabolites and act as miniferritins.

The variability of iron uptake system components among 10 strains from the Stavropol Anti-Plague Institute collection, belonging to 5 of 14 canSNP groups, was expectedly significantly lower than in the entire sample of strains. The phylogenetic reconstruction dendrogram distinguishes 6 genotypes, with strains of closely related canSNP groups A.Br.Ames and A.Br.001/002 belonging to 1 genotype, and the division into the classic main genetic lineages A and B is preserved (Fig. 3).

 

Fig. 3. Phylogenetic reconstruction of the analysis of iron uptake system genes in B. anthracis strains from the collection of pathogenic microorganisms at the Stavropol Anti-Plague Institute.

 

Overall, the frequency of polymorphism variability for hemophores in strains of all B. anthracis lineages was lower than for siderophores, while in strains of B. cereus biovar anthracis lineage C it was approximately the same as for siderophores. Although most strains of lineage A had the same variant of the heme-binding protein Hal as RS, five strains had five variants that differed from each other and from RS due to AAS in different positions. The variants of this protein in strains of lineages B and C differed significantly from RS by the presence of indels; they had two insertions, and the start codon was the triplet TTG. It has been noted that hal expression increases under low iron conditions and that the Sterne strain, which lacks this gene, shows an approximately 100-fold decrease in virulence in mice [21]. It is possible that the demonstrated variability of Hal could affect the virulence of individual strains of lineage A and all strains of lineages B and C.

It appears that hemoglobin heme is the preferred source of iron for B. anthracis. Hemoglobin from hemolyzed erythrocytes is readily available during the extracellular stage of infection and can supply even excessive amounts of heme, whose toxic effect is neutralized by the sensory system that regulates the expression of the heme-detoxifying transporter HrtAB, as well as two immunogenic proteins, Dlp-1 and Dlp-2. Furthermore, the use of hemophores is more energy-efficient than siderophores, since fewer enzymes are involved in their work. It is assumed that at the first intracellular stage of anthrax infection, when hemoglobin heme is unavailable, B. anthracis uses siderophores to absorb iron, which chelate its reserves in phagocytes [19].

Interestingly, IsdC, IsdX1, and IsdX2 have been characterized as potent immunogens during systemic infection when using mice as a biomodel [22].

Conclusion

Certain components of the iron uptake system of B. anthracis are considered virulence or immunogenicity factors, so their characterization will broaden our understanding of the mechanisms of pathogenicity and aid in the development of preventive measures and therapies for anthrax. The variability of the genes and proteins of this system differs among strains of different major genetic lineages and is most pronounced in representatives of the rare C lineage, which have the most polymorphisms, while in strains of the B lineage, the non-ribosomal peptide synthetase necessary for BB biosynthesis is inactive. These features may be associated with the lower adaptive capacity and lower prevalence of the main genetic lineages B and C compared to lineage A. A similar pattern is also shown in the analysis of polymorphisms of genes of other factors of pathogenicity, spore germination, and sporulation. The highest frequency of polymorphisms was observed in strains of B. cereus biovar anthracis, which is explained by the special position of this subspecies, which has a chromosome of classical representatives of B. cereus, significantly different from the chromosome of B. anthracis.

About the authors

Evgeny I. Eremenko

Stavropol Plague Control Research Institute

Author for correspondence.
Email: ejer@mail.ru
ORCID iD: 0000-0002-1117-1185

Dr. Sci. (Med.), Professor, chief researcher, Anthrax laboratory

Russian Federation, Stavropol

Alla G. Ryazanova

Stavropol Plague Control Research Institute

Email: stavnipchi@mail.ru
ORCID iD: 0000-0002-5196-784X

Cand. Sci. (Med.), Head, Anthrax laboratory

Russian Federation, Stavropol

Grigorii A. Pechkovskii

Stavropol Plague Control Research Institute

Email: grigorii.pechkovskii@gmail.com
ORCID iD: 0000-0001-7033-9972

junior researcher, Anthrax laboratory

Russian Federation, Stavropol

Lyudmila Yu. Aksenova

Stavropol Plague Control Research Institute

Email: stavnipchi@mail.ru
ORCID iD: 0000-0002-7744-3112

Cand. Sci. (Med.), senior researcher, Anthrax laboratory

Russian Federation, Stavropol

Olga V. Semenova

Stavropol Plague Control Research Institute

Email: stavnipchi@mail.ru
ORCID iD: 0000-0003-0274-898X

Cand. Sci. (Biol.), researcher, Anthrax laboratory

Russian Federation, Stavropol

References

Supplementary files