College of Agriculture

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As the foundation of the land grant mission at Montana State University, the College of Agriculture and the Montana Agricultural Experiment Station provide instruction in traditional and innovative degree programs and conduct research on old and new challenges for Montana’s agricultural community. This integration creates opportunities for students and faculty to excel through hands-on learning, to serve through campus and community engagement, to explore unique solutions to distinct and interesting questions and to connect Montanans with the global community through research discoveries and outreach.

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Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group AStreptococcusIs Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin
(2019-09) Lei, Benfang; Minor, Dylan; Feng, Wenchao; Jerome, Maria; Quinn, Mark T.; Jutila, Mark A.; Liu, Mengyao
Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.
Pneumococcal surface protein A contributes to secondary Streptococcus pneumoniae infection after influenza virus infection
(2009-08) King, Quinton O.; Lei, Benfang; Harmsen, Allen G.
We compared the growth of Streptococcus pneumoniae mutants with a disruption in the gene for either pneumococcal surface protein A (PspA−), neuraminidase A (NanA−), or hyaluronidase (Hyl−) to that of the parental strain D39 by means of a competitive growth model in mice with and those without prior influenza virus infection. The numbers of total bacteria recovered from mice with prior influenza virus infection were significantly greater than those recovered from mice without prior influenza virus infection. Although the Hyl− and NanA− mutants did not display attenuation in mice with or without prior influenza virus infection, the PspA− mutant exhibited attenuation both in mice with and in mice without prior influenza virus infection. This defect was severe in influenza virus–infected mice, for which growth of the PspA− mutant was 1800-fold lower than that of the parental strain D39. Furthermore, PspA immunization significantly reduced secondary bacterial lung burdens and concentrations of specific markers of lung damage in mice receiving serotypes 2, 3, and 4 pneumococci. Our findings indicate that PspA contributes to secondary S. pneumoniae infection after influenza virus infection and that PspA immunization mitigates early secondary pneumococcal lung infections.
Hypervirulent Group A Streptococcus of Genotype emm3 Invades the Vascular System in Pulmonary Infection of Mice
(2018-06) Lei, Benfang; Minor, Dylan; Feng, Wenchao; Liu, Mengyao
Natural mutations of the two-component regulatory system CovRS are frequently associated with invasive Group A Streptococcus (GAS) isolates and lead to the enhancement in virulence gene expression, innate immune evasion, systemic dissemination, and virulence. How CovRS mutations enhance systemic dissemination is not well understood. A hypervirulent GAS isolate of the emm3 genotype, MGAS315, was characterized using a mouse model of pulmonary infection to understand systemic dissemination. This strain has a G1370T mutation in the sensor kinase covS gene of CovRS. Intratracheal inoculation of MGAS315 led to the lung infection that displayed extensive Gram staining at the alveolar ducts, alveoli, and peribronchovascular and perivascular interstitium. The correction of the covS mutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, the covS mutation allowed MGAS315 to disrupt and degrade the smooth muscle and endothelial layers of the blood vessels, directly contributing to systemic dissemination. It is concluded that hypervirulent emm3 GAS covS mutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination.
Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice
(2017-09) Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Lei, Benfang
Hypervirulent group A streptococcus (GAS) can inhibit neutrophil recruitment and cause systemic infection in a mouse model of skin infection. The purpose of this study was to determine whether platelet-activating factor acetylhydrolase Sse and streptolysin S (SLS) have synergistic contributions to inhibition of neutrophil recruitment and systemic infection in subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. Deletion of sse and sagA in MGAS315 synergistically reduced the skin lesion size and GAS burden in the liver and spleen. However, the mutants were persistent at skin sites and had similar growth factors in nonimmune blood. Thus, the low numbers of Δsse ΔsagA mutants in the liver and spleen were likely due to their reduction in the systemic dissemination. Few intact and necrotic neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and necrotic cells were present at the edge of Δsse mutant infection sites on day 1 and at the edge of and inside Δsse mutant infection sites on day 2. ΔsagA mutant infection sites had massive numbers of and few intact neutrophils at the edge and center of the infection sites, respectively, on day 1 and were full of intact neutrophils or necrotic cells on day 2. Δsse ΔsagA mutant infection sites had massive numbers of intact neutrophils throughout the whole infection site. These sse and sagA deletion-caused changes in the histological pattern at skin infection sites could be complemented. Thus, the sse and sagA deletions synergistically enhance neutrophil recruitment. These findings indicate that both Sse and SLS are required but that neither is sufficient for inhibition of neutrophil recruitment and systemic infection by hypervirulent GAS.
Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of in vitro and in vivo Protease SpeB Expression and Virulence
(2017-01) Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Li, Jinquan; Ishaq, Suzanne L.; Yeoman, Carl J.; Lei, Benfang
Group A Streptococcus (GAS) acquires mutations of virulence regulator CovRS in human and mouse infections that upregulate virulence genes and downregulate protease SpeB. To identify in vivo mutants with novel phenotype, GAS isolates from mouse infection were screened by enzymatic assays for SpeB and platelet-activating factor acetylhydrolase Sse, identifying a new type of variants that had enhanced Sse expression and normal SpeB production (Sse(A+)SpeB(A+)). Sse(A+)SpeB(A+) variants have transcripts levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse(A+)SpeB(A+) isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse(A+)SpeB(A+) isolates also had nonsense mutations or small indels of rocA RocA and CovS mutants have similar enhancement in expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA, but not CovS, do not downregulate speB transcription at stationary growth phase and in subcutaneous infection of mice. RocA and CovS mutations have greater enhancement in expression of hasA than spyCEP in mouse skin infection in comparison with wild type GAS. RocA mutants rank between wild type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infection of mice and exhibit distinct gene expression pattern and virulence from CovS mutants. The findings provide novel information for the understanding of GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.
Spectroscopic Identification of Heme Axial Ligands in HtsA That Are Involved in Heme Acquisition by Streptococcus pyogenes
(2010-04) Ran, Yanchao; Liu, Mengyao; Zhu, Hui; Nygaard, Tyler K.; Brown, Doreen E.; Fabian, Marian; Dooley, David M.; Lei, Benfang
The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV−vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV−vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV−vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a Kd of 40.9 μM but not with H229A HtsA, and cyanide binds to M79A and H229A with Kd of 0.5 and 19.1 μM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.
Transient Weak Protein–Protein Complexes Transfer Heme Across the Cell Wall of Staphylococcus aureus
(2011-09) Villareal, Valerie A.; Spirig, Thomas; Robson, Scott A.; Liu, Mengyao; Lei, Benfang; Clubb, Robert T.
Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus. Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70 000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity “handclasp” complex that juxtaposes their respective 310 helices and β7/β8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.
A periplasmic arsenite-binding protein involved in regulating arsenite oxidation
(2011-12) Liu, Guanghui; Liu, Mengyao; Kim, Eun-Hae; Maaty, Walid S.; Bothner, Brian; Lei, Benfang; Rensing, Christopher; Wang, Gejiao; McDermott, Timothy R.
Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical speciation, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal transduction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a periplasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An ΔaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a KD of 2.4 µM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.
Lipid Oxidation in Trout Muscle Is Strongly Inhibited by a Protein That Specifically Binds Hemin Released from Hemoglobin
(2013-05) Cai, He; Grunwald, Eric W.; Park, Sung Yong; Lei, Benfang; Richards, Mark P.
The recombinant streptococcal protein apoShp can be used as a probe for hemoglobin (Hb) reactivity in fish muscle due to its specific affinity for hemin that is released from Hb at post-mortem pH values. Hemin affinity measurements indicated that apoShp binds hemin released from Hb but not myoglobin (Mb). Hemin affinity of holoShp was higher at pH 5.7 compared to pH 8.0. This may be attributed to enhanced electrostatic interaction of His58 with the heme-7-propionate at lower pH. ApoShp readily acquired hemin that was released from trout IV metHb in the presence of washed cod muscle during 2 °C storage at pH 6.3. This was based on increases in redness in the washed cod matrix, which occurs when apoShp binds hemin that is released from metHb. ApoShp prevented Hb-mediated lipid oxidation in washed cod muscle during 2 °C storage. The prevention of Hb-mediated lipid oxidation by apoShp was likely due to bis-methionyl coordination of hemin that dissociated from metHb. This hexacoordination of hemin appears to prevent peroxide-mediated redox reactions, and there is no component in the matrix capable of dissociating hemin from Shp. ApoShp was also added to minced muscle from rainbow trout (Oncorhynchus mykiss) to examine the degree to which Hb contributes to lipid oxidation in trout muscle. Addition of apoShp inhibited approximately 90% of the lipid oxidation that occurred in minced trout muscle during 9 days of 2 °C storage on the basis of lipid peroxide, hexanal, and thiobarituric acid reactive substances (TBARS) values. These results strongly suggest that Hb is the primary promoter of lipid oxidation in trout muscle.
1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus
(2014-04) Fonner, Brittany A.; Tripet, Brian P.; Lui, Mengyao; Zhu, Hui; Lei, Benfang; Copie, Valerie
Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdBN1). Herein, we report the near complete 1H, 13C and 15N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.

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